EA011419B1 - Multivalent influenza immunogenic composition - Google Patents

Abstract

The present invention relates to influenza vaccine formulations and vaccination regimes for immunising against influenza disease, their use in medicine, in particular their use in augmenting immune responses to various antigens, and to methods of preparation. In particular, the invention relates to multivalent influenza immunogenic compositions comprising an influenza antigen or antigenic preparation thereof from at least two influenza virus strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak, in combination with an oil-in-water emulsion adjuvant.

Description

The present invention relates to influenza vaccine compositions and vaccination regimens for immunization against influenza, their use in medicine, in particular their use in enhancing immune responses to various antigens, and to methods for their manufacture. In particular, this invention relates to multivalent anti-influenza immunogenic compositions comprising an influenza virus antigen or an antigenic preparation thereof from at least two influenza virus strains, wherein at least one strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic, in combination with adjuvant in the form of an oil-in-water emulsion.

State of the art

Influenza virus is one of the most common viruses in the world that affects both people and livestock. Influenza leads to economic burdens, morbidity and even mortality, which are significant.

An influenza virus is an enveloped RNA-containing virus with a particle size of approximately 125 nm in diameter. Essentially, it consists of an internal nucleocapsid or core of ribonucleic acid (RNA) associated with a nucleoprotein surrounded by a viral envelope with a bilayer lipid structure and external glycoproteins. The inner layer of the viral envelope consists mainly of matrix proteins, and the outer layer, mainly of the lipid material of the host. The influenza virus contains two surface antigens - glycoproteins neuraminidase (ΝΑ) and hemagglutinin (HA), which appear in the form of spikes 10-12 nm long on the surface of the particles. These surface proteins, in particular hemagglutinin, determine the antigenic specificity of influenza virus subtypes.

These surface antigens gradually, sometimes quickly, undergo some changes leading to the antigenic diversity of the influenza virus. These antigenic changes, called drift and shift, are unpredictable and can have a strong impact from an immunological point of view, because they ultimately lead to the emergence of new strains of the influenza virus, which allows the virus to elude the immune system and cause well-known, almost annual epidemics.

The influenza virus strains to be included in the influenza vaccine each season are determined by the World Health Organization (WHO) in collaboration with national health services and vaccine manufacturers.

HA is the most important antigen in determining the serological specificity of different strains of the influenza virus. This 75-80 kDa protein contains numerous antigenic determinants, some of which are located in regions whose sequences differ for different strains (strain-specific determinants), while others are located in regions that are common to many HA molecules (common determinants).

Influenza viruses cause epidemics almost every winter, with incidence rates for type A or B viruses up to 40% over a six-week period. Influenza infection causes a variety of painful conditions: from asymptomatic infection through a mild upper respiratory tract infection to severe viral pneumonia. Conventional influenza epidemics cause an increase in pneumonia and lower respiratory tract infections, as evidenced by an increase in hospitalization or mortality. The severity of the disease is determined mainly by the age of the recipient, his immune status and the site of infection.

Elderly people 65 years and older are especially vulnerable, they are associated with 80-90% of all cases of influenza-related death in developed countries. Individuals with chronic diseases are also more likely to experience such complications. Young children can also suffer from a serious illness. Therefore, these groups are especially in need of protection. In addition to these risk groups, health services also recommend vaccinating healthy adults in contact with older people.

Vaccination plays a critical role in managing annual influenza epidemics. Currently available influenza vaccines are either inactivated or live attenuated influenza vaccines (Di-vaccines). The composition of inactivated Dc vaccines includes three possible forms of antigenic preparation: inactivated whole virus, subvirions, when purified virus particles are destroyed by detergents or other reagents to solubilize the lipid membrane (the so-called split vaccine (split vaccine)), or purified HA and ΝΑ ( subunit vaccine). These inactivated vaccines are administered intramuscularly (i / m) or intranasally (i / n).

All types of influenza vaccines are usually trivalent vaccines. As a rule, they contain antigens of two strains of influenza A virus and one strain of influenza B. The standard 0.5 ml injection dose in most cases contains 15 μg of the antigenic component of hemagglutinin from each strain, as measured by simple radial immunodiffusion (8JE) (1.M Aob s1 a1 .: Appgousb kshd1s gab1a1 shshshpobDDikyup 1se1shk | is Dog 111s akau οί shPispha yastaddshshsh aiDdei: abarShopp Dog rosspusyuyuyuuuuuuuuuuuuuuuuuuuuuuuu.

- 1 011419

237-247; Hm. \ Woo s! A1., 1p! stpa! jupa1 sopaogaNus Chibu οί 81pd1s gab1a1 b | GGichop apb ppppshpos1ss1gor1yugschk 1ss11pk. 1. BU1. 8! Apb. 9 (1981) 317-330).

Currently available influenza vaccines are considered safe for all age groups (Os EopaU!! A1., 1999, Wassts, 17, 3094-3101). However, there is little evidence that existing influenza vaccines work in young children under the age of 2 years. In addition, the vaccine efficacy ratios cited in the prevention of a typical confirmed influenza disease are 23–72% for older people, which is significantly lower than the 60–90% efficacy ratios for younger adults (SosasP. 1994, 1. At. Akkos., 21, 166-1665; Sgokk, 1995, App. 1p! Group Msb. 123, 523-527). The effectiveness of the influenza vaccine has been shown to correlate with the serum titers of hemagglutination inhibiting antibodies (ΗΙ) for this viral strain, and several studies have found that older people show lower тит titers after immunization with influenza virus than younger adults (Migakko , 2002, Expsptsp1a1 dstop! O1o§u, 37, 427-439).

Therefore, there is still a need for new vaccines with increased immunogenicity. The composition of a vaccine antigen with highly effective adjuvants is a possible approach to enhancing the immune responses to subvirion antigens.

A subunit influenza vaccine with an ME59 adjuvant in the form of an oil-inlet emulsion is commercially available and has demonstrated its ability to induce a higher titer of antibodies than the titer obtained using a non-adjuvant subunit vaccine (Os Eopa! O!! A1., 1999, Wassts, 17 , 3094-3101). However, in a later publication, the same vaccine did not show an improved profile compared to the non-adjuvant split vaccine (Rhd-Wabsta! A1., 2004, Wassts, 23, 283-289).

By way of explanation, influenza viruses that are related to the viruses of a previous epidemic circulate during interpandemic periods. These viruses spread among people with different levels of immunity to infections from a previous period of life. Such circulation over a period of usually 2–3 years stimulates the selection of new strains that have changed sufficiently to cause an epidemic again in the general population; this process is referred to as antigenic drift. Drifting options can have different effects in different communities, regions, countries or continents over any one year, however, in a few years their total impact will often be similar. In other words, an influenza pandemic occurs when a new influenza virus appears that the human population is not immune to. Conventional influenza epidemics cause an increase in pneumonia and lower respiratory tract infections, as evidenced by an increase in hospitalization or mortality rates. Older people or people with chronic illnesses will almost certainly be prone to such complications, and young children may also suffer a serious illness.

At unpredictable intervals, new influenza viruses emerge with a subtype of the key surface antigen, hemagglutinin, which is completely different from the subtype in the strains circulating in the previous season. And here the obtained antigens may differ by 2050% from the corresponding protein strains that previously circulated among people. This can lead to the escape of the virus from the immunity of the population and the establishment of pandemics. This phenomenon is called antigenic shift. It is believed that at least in the past, pandemics occurred when an influenza virus from different species, such as avian influenza virus or swine influenza virus, crossed the species barrier. If such viruses have the ability to spread from subject to subject, then they can spread around the world in the range from several months to a year, which leads to a pandemic. For example, in 1957. (pandemic of Asian flu (Ak1a E1i)) viruses of the Η2Η2 subtype replaced the Η1Ν1 viruses that have been circulating in the human population since at least 1918, when this virus was first isolated. H2 HA and N2 NA underwent antigenic drift between 1957 and 1968. until the NA was supplanted in 1968. (pandemic of the Hong Kong flu (Iopd-Kopd E1i)) as a result of the emergence of the subtype of the influenza virus Η3 ,2, after which N2 NA continued to drift along with H3 ON Schakadta s! A1., 1991, Er1bstu1. 1pGss !. 106, 383-395).

The properties of an influenza virus strain that make it capable of causing an outbreak of a pandemic are as follows: it contains new hemagglutinin compared to the hemagglutinin of currently circulating strains, which may or may not be accompanied by a change in the subtype of neuraminidase; he has the ability to be transmitted horizontally in the human population; and it is pathogenic to humans. The new hemagglutinin may be hemagglutinin that has not been manifested in the human population for an extended period of time, possibly for several decades, such as H2. Or it may be hemagglutinin that has not circulated in the human population before, such as H5, H9, H7 or H6, which are found in birds. In any case, the majority, or at least a large part of the population, or even the entire population, has not encountered this antigen before and is immunologically naive in relation to it.

There is still a need for improved influenza vaccines, especially in cases

- 011419 pandemic influenza pandemics for the elderly.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a multivalent anti-influenza immunogenic composition comprising an influenza virus or antigenic preparation of at least two influenza virus strains, wherein at least one strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic, in combination with an adjuvant in in the form of an oil-in-water emulsion, wherein said adjuvant in the form of an oil-in-oil emulsion contains a metabolizable oil, a sterol and an emulsifier. Preferably, said sterol is alpha tocopherol.

Suitable pandemic strains include, but are not limited to, Η5Ν1, Η9Ν2, Η7Ν7, Η2Ν2 and Η1Ν1.

In another aspect, the invention provides a method for preparing an influenza immunogenic composition for a pandemic situation, comprising mixing an influenza virus antigen or antigenic preparation from at least two strains of influenza virus, at least one of which is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic, with an oil-in-water emulsion as defined hereinabove.

In a third aspect, an immunogenic composition, as defined herein, is provided for use in medicine.

In another aspect, the use of: (a) an antigen of an influenza virus or its antigenic preparation, and (b) an adjuvant in the form of an oil-in-water emulsion in the manufacture of an immunogenic composition for inducing at least one of the following: 1) enhanced ί'ί4 T -cellular immune response, 2) an enhanced B-cell secondary immune response against the indicated viral antigen or antigenic composition in humans, preferably in an individual or population with weakened immunity, such as adults or elderly people from a group of high risk, which is preferred. Preferably, the immunogenic composition is as defined herein.

In addition, it is proposed the use of influenza virus or its antigenic preparation and adjuvant in the form of an oil-in-water emulsion in the manufacture of an immunogenic composition, as defined herein, for vaccinating elderly people against influenza.

In a specific embodiment, the immunogenic composition is capable of inducing both an enhanced ί'Ό4 T-cell immune response and an enhanced B-cell secondary immune response compared to that obtained using a non-adjuvant antigen or antigenic composition.

In another embodiment, the use of the influenza virus or its antigenic preparation in the manufacture of an immunogenic composition for revaccinating people pre-vaccinated with a polyvalent anti-influenza immunogenic composition comprising an influenza virus antigen or antigenic preparation of at least two influenza virus strains, wherein at least one strain is associated, is provided. with an outbreak of a pandemic, or may be associated with an outbreak of a pandemic, in combination with an adjuvant in the form of an oil-in-water emulsion as defined herein NII.

In a specific embodiment, the composition used for revaccination may be adjuvantless or may contain an adjuvant, in particular an adjuvant in the form of an oil-in-water emulsion. In another specific embodiment, the immunogenic revaccination composition comprises an influenza virus or antigenic preparation thereof that has common CE4 T cell epitopes with the influenza virus or antigenic preparation of the virus used for the first vaccination.

Preferably, revaccination is carried out in subjects who have been vaccinated against influenza in the previous season. Typically, revaccination is carried out at least 6 months after the first vaccination, preferably after 8-14 months, more preferably after about 10-12 months.

Preferably, said oil-in-water emulsion comprises a metabolizable oil, alfatocopherol and an emulsifier. In another specific embodiment, said oil-in-water emulsion adjuvant contains at least one metabolizable oil in an amount of from 0.5 to 20% of the total volume and has oil droplets whose diameters are at least 70% in intensity are less than 1 micron.

In a further aspect of the present invention, there is provided the use of an antigen or antigenic preparation from a first influenza virus strain in the manufacture of an immunogenic composition, as defined herein, for protecting against influenza virus infection caused by a variant influenza virus strain.

In another aspect, a method is provided for vaccinating an individual or a weakened immune population, such as adults or elderly people from a high-risk group, with an influenza immunogenic composition containing an influenza virus antigen or an antigenic preparation of at least two influenza virus strains, where at least one strain is associated with an outbreak of a pandemic, or may be associated with an outbreak of a pandemic, in combination with an adjuvant in the form of an oil-in-water emulsion, as defined herein.

In yet another embodiment according to the invention, a method for revaccinating people,

- 3 011419 pre-vaccinated with a polyvalent anti-influenza immunogenic composition containing an influenza virus antigen or antigenic preparation of at least two influenza virus strains, where at least one strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic, in combination with an adjuvant in in the form of an oil-in-water emulsion comprising administering to a specified person an immunogenic composition containing an influenza virus, either adjuvant or non-adjuvant.

In another embodiment, a method is provided for vaccinating a human population or an individual against one strain of influenza virus, followed by revaccinating said person or population against a variant strain of influenza virus, comprising administering to said person: (1) a first composition comprising an influenza virus or antigenic preparation thereof from a first strain influenza virus and adjuvant in the form of an oil-in-water emulsion, and (2) a second immunogenic composition comprising a variant strain of influenza virus of said first virus strain Rippey. In a specific embodiment, said first strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. In another specific embodiment, said variant strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic.

Other aspects and advantages of the present invention are described further in the following detailed description of its preferred embodiments.

Description of figures

FIG. 1. Size distribution of oil droplets for an 8B62 oil-in-water emulsion, measured by PC8 (photon correlation spectroscopy). In FIG. 1A shows the size measurement results for 8В62 batch 1023 using Ma1uegi ΖοΙαδίζοΓ 3000Н8: А corresponds to a dilution of 1/10000 (protocols 22-24) (analysis according to the Soiyi method and using the adapted optical model 1.5 / 0.01); B corresponds to a dilution of 1/20000 (protocols 28-30) (analysis according to the Soiyi method and using the adapted optical model 1.5 / 0.01). In FIG. 1B shows a schematic illustration of the data of protocol 22 (upper part) and protocol 23 (lower part) in intensity.

FIG. 2. Schematic illustration of the preparation of bulk form MRI.

FIG. 3. Schematic illustration of the preparation of adjuvant A803 + MPI.

FIG. 4. Clinical trial Exp1o-P1i-001. CE4 T-cell response to the cleaved influenza virus antigen (01 = first quartile, O3 = third quartile).

FIG. 5. Clinical trial Exp1o-P1i-001. SE8 T-cell response to the cleaved influenza virus antigen (01 = first quartile, O3 = third quartile).

FIG. 6. Clinical trial Exp1o-P1i-001. Cross-reactive CE4 T-cell response to cleaved influenza virus antigen after P1iapx + A803 vaccination.

FIG. 7. Clinical trial Exp1o-P1i-001. B-cell secondary immune response after vaccination.

FIG. 8. Clinical trial Exp1o-P1i-002. CE4 T-cell response against a split influenza virus antigen after booster vaccination.

FIG. 9. Clinical trial Exp1o-P1i-002. Anti-H1 titers after revaccination.

FIG. 10. Research I on ferrets. Temperature monitoring (priming and control infection). FIG. 10A corresponds to priming, FIG. 10B corresponds to the challenge.

FIG. 11. Research I on ferrets. Viral shedding.

FIG. 12. Study II on ferrets. Temperature monitoring (priming and control infection). FIG. 12A corresponds to priming, FIG. 12B corresponds to control infection.

FIG. 13. Study II on ferrets. Viral shedding.

FIG. 14. Study II on ferrets. W-titers to Η3Ν2 A / Raiata (vaccine strain) (Fig. 14A) and to Η3Ν2 AJuott (strain for experimental infection) (Fig. 14B).

FIG. 15. Study on mice. The frequency of occurrence of CE4 T cells in primed C57BB / 6 mice as a result of using the whole inactivated virus as a repeated stimulating antigen (7th day after immunization).

FIG. 16. Research on mice. The frequency of occurrence of CE8 T cells in primed C57BP6 mice as a result of using the whole inactivated virus as a repeated stimulating antigen (7th day after immunization).

FIG. 17. Research on mice. The frequency of occurrence of CE4 (upper part) and SE8 (lower part) of T cells in C57BP6 mice primed with heterologous strains resulting from the use of a whole inactivated virus as a repeated stimulating antigen (7th day after immunization).

FIG. 18. Clinical trial in humans. B-Cellular secondary immune response after vaccination of the elderly with P1iapkh, P1iapkh + A803, P1iapkh + A803 + MPB (difference between the conditions before and after vaccination (rge and rpoch)).

FIG. 19. Research III on ferrets. Temperature monitoring before and after challenge.

FIG. 20. Research III on ferrets. Viral shedding before and after challenge.

FIG. 21. Study III on ferrets. W-titers to Η3Ν2 A / ^ Wattide (vaccine strain).

- 4 011419

FIG. 22. Study III on ferrets. ΗΙ-titers to Η3Ν2 A / Rapata (strain for experimental infection).

FIG. 23. Clinical trial in humans. ΗΙ-titers (SMT; geometric mean titers) on the 21st, 90th and 180th days after vaccination (persistence).

FIG. 24. Clinical trial in humans. CO2-response according to the test, all two per antigen pool on the 21st, 90th and 180th days after vaccination (persistence).

FIG. 25. Clinical trial in humans. ΗΙ-Titers in a clinical trial with A803 + MPB revaccination as compared to E1iapch.

FIG. 26. Clinical trial in humans. CM1 (cell-mediated immunity) according to the CE4 response according to the test, all two per pool of antigens on days 0 and 21.

FIG. 27. Clinical trial in humans using A803 + MPI in two concentrations. ΗΙTitles on the 0th and 21st day.

FIG. 28. Clinical trial in humans using A803 + MPI in two concentrations. Reactogenicity.

Detailed description

The authors of the present invention have found that an influenza composition containing an influenza virus or antigenic preparation thereof together with an adjuvant in the form of an oil-in-water emulsion containing a metabolizable oil, a sterol such as alpha-tocopherol, and an emulsifier capable of enhancing ί.Ό4 T -cellular immune response and / or B-cell secondary immune response against the specified antigen or antigenic composition in humans compared with the response obtained using the adjuvant virus or its antigenic preparation. Compositions supplemented with an oil-in-water emulsion adjuvant, as defined herein, will primarily be used to induce a CE4 T cell response against influenza virus capable of detecting influenza virus epitopes represented by class II MIS molecules (major histocompatibility complex) . In this work, the authors of the present invention found that, as a target, they effectively act on the cell-mediated immune system in order to enhance the immune response against homologous and drifting strains of the influenza virus (after vaccination and infection).

Adjuvant anti-influenza compositions according to the invention have several advantages:

1) increased immunogenicity: they will restore the weak immune response in the elderly (over the age of 50 years, usually over the age of 65) to the levels observed in young people (antibody and / or T-cell responses);

2) an improved cross-immunity profile: increased cross-immunity against various (drifting) strains of the influenza virus;

3) they will also reduce the dosage of antigen to obtain a similar response, thereby guaranteeing increased potential in emergency situations (pandemics, for example).

Compositions for use in the present invention were able to provide better serological protection against influenza virus after revaccination, as evaluated in a group of people who meet the established correlation of protection against influenza. In addition, the composition for use in the present invention is also capable of inducing a tendency to a higher B-cell secondary immune response after the first vaccination of a human subject and to a higher humoral response after revaccination compared to the non-adjuvant composition.

In addition, the inventors were able to show that the claimed adjuvant composition was able to not only induce, but also maintain protective levels of antibodies against all three strains present in the vaccine, in a greater number of individuals compared to levels obtained using a non-adjuvant composition (see table 43, for example).

Thus, in yet another embodiment, the claimed composition is capable of guaranteeing a persistent immune response against an influenza-associated disease. In particular, persistence is understood to mean a W-antibody response that is able to satisfy regulatory criteria at least 3 months, preferably at least 6 months after vaccination. In particular, the claimed composition is capable of inducing protective antibody levels in more than 70% of individuals, preferably in more than 80% of individuals or preferably in more than 90% of individuals for at least one strain of influenza virus, preferably for all strains present in the vaccine, at least in 3 months. In a specific aspect, protective antibody levels of more than 90% are obtained at least 6 months after vaccination against at least one, preferably two or all of the strains present in the vaccine composition.

Strains and antigens of influenza virus

The influenza virus or its antigenic preparation for use according to the present invention may be a split influenza virus or an antigenic preparation of this split virus. In an alternative embodiment, the influenza virus preparation may contain another type of antigen of an inactivated influenza virus, such as an inactivated whole virus, or purified HA and NA (subunit vaccine), or an influenza virosome. In yet another embodiment, the influenza virus may be a preparation of live attenuated influenza virus.

- 5 011419

The cleaved influenza virus or antigenic preparation of this cleaved virus for use according to the present invention is, respectively, an inactivated viral preparation in which the viral particles are destroyed by detergents or other reagents for solubilizing the lipid membrane. The split virus or antigenic preparations of this split virus, respectively, are made by fragmenting the whole influenza virus, either infectious or inactivated, using organic solvents or detergents in solubilizing concentrations and then removing all or most of the solubilizing agent and some or most of the viral lipid material . By an antigenic preparation of such a cleaved virus is meant a cleaved viral preparation that can be purified to some extent compared to a cleaved virus, while preserving most of the antigenic properties of the cleaved viral components. For example, in the case of production on chicken embryos, the cleaved virus can be purified from impurity proteins of chicken embryos or, in the case of production in cell culture, the cleaved virus can be purified from impurities of the host cells. A cleaved viral antigenic preparation may contain cleaved viral antigenic components of more than one viral strain. Vaccines containing cleaved virus (referred to as the influenza split vaccine) or cleaved viral antigenic preparations usually contain residual matrix proteins, and nucleoproteins, and sometimes lipids, as well as membrane envelope proteins. Such antiviral split vaccines will usually contain most or all of the viral structural proteins, although the proportions in which they are present in the whole virus are not necessary.

Alternatively, the influenza virus may be presented in the form of a whole-virion vaccine. It could be proved that this is an advantage relative to the cleaved viral vaccine in a pandemic situation, since it avoids the uncertainty in the possibility of successfully producing the cleaved viral vaccine against a new strain of influenza virus. For some strains, the traditional detergents used to prepare the cleaved virus can damage the virus and render it unsuitable for use. Despite the fact that it is always possible to use other detergents and / or to develop another method for preparing a split vaccine, this will take time, which may not be enough in a pandemic situation. In addition to a higher degree of certainty when using the whole-virion approach, the production capacity of vaccines is also higher than in the case of a split virus, since significant amounts of antigen are lost during the additional purification steps necessary to produce a suitable split vaccine.

In another embodiment, the influenza virus preparation is in the form of a purified subunit flu virus. Subunit influenza vaccines usually contain two major envelope proteins: HA and ΝΑ, and may have an additional advantage over whole-virion vaccines, since they are usually less reactogenic, in particular for young vaccinated subjects. Subunit vaccines can be obtained either recombinantly or purified from disrupted viral particles.

In another embodiment, the influenza virus preparation is in the form of a virosome. Virosomes are spherical unilamellar vesicles that contain functional viral envelope glycoproteins HA and ΝΑ in an authentic conformation, intercalated into the phospholipid bilayer membrane of virosomes.

The specified influenza virus or its antigenic preparation can be obtained from chicken embryos or tissue cultures.

For example, an influenza virus antigen or antigenic preparations of the invention can be prepared in a conventional manner using eggs with a developing embryo by growing the influenza virus in eggs and purifying the collected allantoic fluid. Eggs can be collected in large quantities in a short time. Alternatively, they (antigens) can be obtained using any of the new tissue culture production methods for growing the virus or expressing recombinant surface influenza virus antigens. Suitable cell substrates for growing the virus include, for example, dog kidney cells, such as ISKCON or ISKCON clone cells, MESK-like cells, monkey kidney cells, such as ASMK cells (African green monkey kidney cells), including Huego cells, suitable pig cell lines or any other type of mammalian cell suitable for the production of influenza virus for the manufacture of vaccines. Suitable cell substrates also include human cells, for example, ISS-5 cells. Suitable cell substrates are not limited to cell lines; for example, primary cells such as chicken embryonic fibroblasts and bird cell lines are also included.

An influenza virus antigen or antigenic preparation thereof can be obtained by any of a variety of commercially used methods, for example, using the split-Di method described in patents Nos. 300833 and 211444, incorporated herein by reference. Traditionally, splitDi was prepared using a solvent / detergent treatment such as tri-n-butyl phosphate or diethyl ether in combination with Teteep ™ (known as twin ether cleavage), and this method is still used in some production plants. Other currently used

- 6 011419 cleavage agents include detergents, or proteolytic enzymes, or salts of bile acids, for example sodium deoxycholate, as described in patent No. 155875, incorporated herein by reference. Detergents that can be used as cleavage agents include cationic detergents, for example cetyltrimethylammonium bromide (CTAB), other ionic detergents, for example lauryl sulfate, taurodeoxycholate, or non-ionic detergents as described above, including Triton X-100 (for example, the method described in Ipa c1 a1., 2000, Vu1o§1sa18, 28, 95-103) and Triton N-101, or a combination of any two or more detergents.

A method for preparing a split vaccine may include many different stages of filtration and / or other stages of separation, such as stages of ultracentrifugation, ultrafiltration, zone centrifugation and chromatography (e.g., ion exchange), in various combinations, and possibly an inactivation step, for example, by heating, formaldehyde or β-propiolactone or UV, which can be carried out before or after cleavage. The splitting method can be carried out in the form of a batch, continuous or semi-continuous method. A preferred cleavage method and method for purifying an immunogenic split composition is described in νθ 02/097072.

Preferred split-Di-vaccine antigenic preparations according to the invention contain a residual amount of Tween 80 and / or Triton X-100 remaining after the production method, although they can be added or brought up after preparation of the split antigen. Preferably, when both are present: Tween 80 and Triton X-100.

Preferred ranges for the final concentrations of these non-ionic surfactants in the vaccine dose are:

Tween 80: 0.01-1%, more preferably about 0.1% (v / v);

Triton X-100: 0.001-0.1 (% w / v), more preferably 0.005-0.02% (w / v).

In a specific embodiment, the final concentration of Tween 80 varies from 0.045 to 0.09% (w / v). In another specific embodiment, the antigen is provided as a 2-fold concentrated mixture in which the Tween 80 concentration varies from 0.045 to 0.2% (w / v) and which must be diluted 2 times after preparation of the final composition with an adjuvant (or buffer in the control composition).

In another specific embodiment, the final concentration of Triton X-100 varies from 0.005 to 0.017% (w / v). In another specific embodiment, the antigen is provided as a 2 times more concentrated mixture, the concentration of Triton X-100 in which varies from 0.005 to 0.034% (w / v) and which must be diluted 2 times to prepare the final composition with adjuvant (or with buffer in the control composition).

Preferably, an influenza virus preparation is prepared in the presence of a low level of thiomersal, or preferably in the absence of thiomersal. Preferably, the resulting influenza virus preparation is stable in the absence of organomercury preservatives, in particular the preparation does not contain any residual thiomersal. An influenza virus preparation, in particular, contains a hemagglutinin antigen stabilized in the absence of thiomersal or at low levels of thiomersal (usually 5 μg / ml or less). Specifically, stabilization of the influenza B virus strain is carried out using an alpha-tocopherol derivative such as alpha-tocopherol succinate (also known as vitamin E succinate, i.e., UE8). Such preparations and methods for their preparation are disclosed in νθ 02/097072.

The preferred composition contains three inactivated cleaved virion antigens prepared from WHO recommended influenza virus strains of the corresponding season.

Preferably, the influenza virus or its antigenic preparation and the oil-in-water emulsion adjuvant are contained in the same container. It is designated as an approach in one bottle. Preferably, such a vial is a pre-filled syringe. In an alternative embodiment, the influenza virus or its antigenic preparation and an oil-water emulsion adjuvant are contained in separate containers or vials and are rapidly mixed before or during administration to a subject. It is designated as an approach in two bottles. As an example, when the vaccine is a 2-component vaccine with a total dose volume of 0.7 ml, concentrated antigens (for example, concentrated trivalent inactivated cleaved virion antigens) are in one bottle (335 μl) (antigen container), and pre-filled the syringe contains an adjuvant (360 μl) (container for adjuvants). At the time of injection, the contents of the vial containing concentrated trivalent inactivated cleaved virion antigens are removed from the vial using a syringe containing adjuvant, followed by gentle mixing of the contents of the syringe. Before injection, the used needle is replaced with an intramuscular injection needle and the volume is adjusted to 530 μl. One dose of reconstituted adjuvanted influenza vaccine candidate corresponds to 530 μl.

According to the present invention, at least one strain of influenza virus in a multivalent immunogenic composition, as defined herein, is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. Such a strain may also be referred to below as pandemic strains. In particular, when the vaccine is a multivalent, for example a divalent, or trivalent, or tetravalent vaccine, then at least one strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. Suitable strains are

- 7 011419 by themselves, but are not limited to this, Η5Ν1, Η9Ν2, Η7Ν7, Η2Ν2 and Η1Ν1.

Conveniently, when the specified influenza virus or its antigenic preparation is polyvalent, for example divalent, or trivalent, or tetravalent. Preferably, the influenza virus or antigenic preparation thereof is trivalent or tetravalent having an antigen from three different strains of influenza virus, wherein at least one strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic.

The properties of an influenza virus strain that make it capable of causing an outbreak of a pandemic are as follows: it contains new hemagglutinin compared to the hemagglutinin of the currently circulating strains; he has the ability to be transmitted horizontally in the human population; and it is pathogenic to humans. The new hemagglutinin may be hemagglutinin that has not been manifested in the human population for an extended period of time, possibly for several decades, such as H2. Or it may be hemagglutinin that has not circulated in the human population before, such as H5, H9, H7 or H6, which are found in birds. In any case, the majority, or at least a large part of the population, or even the entire population, has not encountered this antigen before and is immunologically naive in relation to it.

Typically, some groups have an increased risk of getting the flu in a pandemic situation. Elderly people, chronically ill people and young children are especially susceptible, however, many young and clearly healthy people are also at risk. As for the H2 influenza virus, part of the population born after 1968 has an increased risk. It is important for these groups to get effective protection as quickly and easily as possible.

Another group of people who are at high risk are travelers. People now travel more than ever before, and the regions where most of the new viruses are emerging, China and Southeast Asia, have become popular travel destinations in recent years. This change in travel patterns allows new viruses to spread across the globe in a few weeks, not months or years.

Thus, for these groups of people there is a special need for vaccination to protect against influenza in a pandemic situation or a possible pandemic situation. Suitable strains are Η5Ν1, Η9Ν2, Η7Ν7, Η2Ν2 and Η1Ν1, but are not limited to this.

Perhaps the composition may contain more than three valencies, for example, two non-pandemic strains plus a pandemic strain. Alternatively, the composition may contain three pandemic strains. Preferably, the composition comprises three pandemic strains.

Oil-in-Water Emulsion Adjuvant

The adjuvant composition of the invention contains an adjuvant in the form of an oil-in-water emulsion, preferably said emulsion contains metabolizable oil in an amount of from 0.5 to 20% of the total volume and has oil droplets whose diameter is at least 70% in intensity, is less than 1 micron.

In order for any oil-in-water composition to be suitable for administration to humans, the oil phase of the emulsion system must contain a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as capable of conversion through metabolism (illustrated by Dorland (Er1appb) Medical Dictionary (Mob1ca1 Er | s1yupagu. Ν.Β. §aibet8 Sotrapu, 25111 οάίΐίοη (1974)). The oil may be any vegetable oil, fish oil, animal oil or synthetic oil that is not toxic to the recipient and is capable of conversion through metabolism. Nuts, grains and cereals are common sources of vegetable oils. Synthetic oils are also part of this invention, and they may include commercially available oils such as ΝΕΟΒΕΕ® and others. A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil that is found in large quantities in shark liver oil and in smaller quantities in olive oil, wheat seed oil, rice bran oil and yeast, and it is a particularly preferred oil for use in this invention. Squalene is a metabolizable oil because it is an intermediate in cholesterol biosynthesis (Megsk 1pbeh, 10th edition, entry No. 8619).

Oil-in-water emulsions are well known in the art and are believed to be useful as adjuvant compositions (ΕΡ 399843; νΟ 95/17210).

Accordingly, the metabolizable oil is present in an amount of from 0.5 to 20% (final concentration) of the total volume of the immunogenic composition, preferably in an amount of 1.0 to 10% of the total volume, preferably in an amount of 2.0 to 6.0% of the total volume.

In a specific embodiment, the metabolizable oil is present in a final amount of about 0.5, 1, 3.5, or 5% of the total volume of the immunogenic composition. In another specific embodiment, the metabolizable oil is present in a final amount of 0.5, 1, 3.57 or 5% of the total volume of the immunogenic composition.

- 8 011419

Preferably, the oil-in-water emulsion systems of the present invention have a small, in the submicron range, oil droplet size. Accordingly, the droplet sizes will be in the range of 120-750 nm, sizes from 120 to 600 nm in diameter are more preferable. Most preferably, when the oil-in-water emulsion contains oil droplets whose diameter is at least 70% in intensity, less than 500 nm, more preferably at least 80% in intensity, the diameter is less than 300 nm, more preferably at least at least 90% in intensity, the diameter lies in the range of 120-200 nm.

Oil droplet size, i.e. the diameter according to the present invention is given in intensity. There are several ways to determine the diameter of oil droplets by intensity. The intensity is measured using a size distribution apparatus, respectively, using dynamic light scattering, such as a Model ΖοΙαδίζοτ 4000 or preferably a Model ΖοΙαδίζοτ 3000Н8. A detailed procedure is given in Example A.2. The first possibility is to determine the average diameter ζ (ΖΛΌ) using dynamic light scattering (PC8 - photon correlation spectroscopy); this method further provides a polydispersity index (ΡΌΙ). and like ΖΛΌ. so and ΡΌΙ are calculated using the cumulative analysis algorithm. For these quantities, knowledge of the refractive index of the particle is not required. The second method consists in calculating the diameter of the oil droplet by determining the size distribution of all particles using another algorithm, either Co1sh, or ΝΝΡ8 (non-negative least squares), or the automated Layout algorithm (the default algorithm specified by the device for size distribution). In most cases, when the refractive index of a particle of a complex composition is not known, only the intensity distribution is considered, and if necessary, the average value of the intensity resulting from this distribution.

The oil-in-water emulsion according to the invention contains sterol. Sterols are well known in the art, for example, cholesterol is well known, and it is described, for example, in Megsk 1ibekh, 11th edition, p. 341, as a naturally occurring sterol found in animal fat. Other suitable sterols include β-sitosterol, stigmasterol, ergosterol, alpha-tocopherol and ergocalciferol. Preferably, said sterol is present in an amount of from 0.01 to 20% (w / v) of the total volume of the immunogenic composition, preferably in an amount of from 0.1 to 5% (w / v). Preferably, when the sterol is cholesterol, then it is present in an amount of from 0.02 to 0.2% (w / v) of the total volume of the immunogenic composition, more preferably in an amount of 0.02% (w / v) in the volume of the vaccine dose of 0.5 ml, or 0.07% (w / v) in the volume of the vaccine dose of 0.5 ml, or 0.1% (w / v) in the volume of the vaccine dose of 0.7 ml .

Accordingly, the sterol is alpha-tocopherol or a derivative thereof, such as alpha-tocopherol succinate. Preferably, alpha-tocopherol is present in an amount of 0.2 to 5.0% (v / v) of the total volume of the immunogenic composition, more preferably in an amount of 2.5% (v / v) of the vaccine dose 0 , 5 ml, or 0.5% (v / v) in the volume of the vaccine dose of 0.5 ml, or 1.71.9% (v / v), preferably 1.8% in the volume of the vaccine dose 0 , 7 ml. For clarity, the concentrations given in v / v can be converted to the concentration in w / v. by using the following conversion factor: a concentration of alpha-tocopherol of 5% (v / v) is equivalent to a concentration of alpha-tocopherol of 4.8% (w / v).

The oil-in-water emulsion may further comprise an emulsifier. The emulsifier may be present in an amount of 0.01-5.0% (w / w) by weight of the immunogenic composition, preferably present in an amount of 0.1-2.0% (w / w) by weight. A preferred concentration is 0.51.5% (w / w) by weight of the total composition.

The emulsifier, respectively, can be a polyoxyethylene sorbitan monooleate (Tween 80). In a specific embodiment, a 0.5 ml vaccine dose volume contains 1% (w / w) Tween 80, and a 0.7 ml vaccine dose volume contains 0.7% (w / w) Tween 80. In another specific embodiment the concentration of Tween 80 is 0.2% (w / w).

An oil-in-water emulsion adjuvant can be used with other adjuvants or immunostimulants, and therefore an important embodiment of the present invention is an oil-in-water composition comprising squalene or another metabolizable oil, alpha-tocopherol and Tween 80. Emulsion An oil-in-water type may also contain an edge of 85 and / or lecithin. Typically, an oil-in-water emulsion will contain 2-10% squalene of the total immunogenic composition, 2-10% alpha-tocopherol and 0.3-3% Tween 80, and can be prepared according to the procedure described in XV O 95 / 17210. Preferably, the ratio of squalene: alpha-tocopherol is equal to or less than 1, as this provides a more stable emulsion. 8pai 85 (polyoxyethylene sorbitan trioleate) may also be present, for example, at a level of 1%.

Immunogenic properties of the immunogenic composition used for the first vaccination of the present invention

In the present invention, the multivalent anti-influenza composition is capable of inducing an enhanced CE4 T cell immune response against at least one of the components of the multicomponent antigen (s) or antigenic composition compared to the CE4 T cell immune

- 9 011419 by the answer obtained using the appropriate composition that is adjuvant-free, i.e. does not contain any exogenous adjuvant (also referred to in this description as a simple (p1ax) composition). In a specific embodiment, said enhanced CE4 T-keto immune response is a response against a pandemic strain of influenza virus.

By an enhanced CE4 T cell immune response is meant that a higher CE4 response is obtained in a human patient after administration of an adjuvant immunogenic composition compared to an immune response obtained after administration of the same composition without an adjuvant. For example, a higher CE4 T-cell response is obtained in a human patient after administration of an immunogenic composition containing an influenza virus or antigenic preparation together with an adjuvant in the form of an oil-in-water emulsion containing a metabolizable oil, alpha-tocopherol and emulsifier, by compared with the response induced after the introduction of the immunogenic composition containing the influenza virus or its antigenic preparation. Such a composition will advantageously be used to induce a CE4 T cell response against influenza virus capable of detecting influenza virus epitopes represented by MHC class II molecules.

Preferably, when the specified immunological response induced by the adjuvanted composition of the cleaved influenza virus for use in the present invention is higher than the immunological response induced by any other non-adjuvant traditional influenza vaccine, such as subunit influenza vaccine or whole virus influenza vaccine.

In particular, but not exclusively, said enhanced CE4 T-cell immune response is obtained from an immunologically non-primed patient, i.e. a patient who is seronegative for the specified influenza virus or antigen. This seronegativity may be due to the fact that the specified patient never encountered such a virus or antigen (the so-called naive patient) or, alternatively, did not give a reaction to the specified antigen after meeting with him. Preferably, said enhanced CE4 T cell immune response is obtained in a subject with a weakened immune system, such as an elderly person, usually at least 50 years old, usually 65 years old or older, or an adult under 65 years old at high risk medical condition (adult at high risk), or a child under the age of 2 years.

Enhanced CE4 T-cell immune response can be assessed by measuring the number of cells producing any of the following cytokines:

cells producing at least two different cytokines (CE40b, 1b-2 (interleukin-2), ΙΡΝγ (interferon γ), ΤΝΡα (tumor necrosis factor alpha));

cells producing at least CE40b and another cytokine (1b-2, ΤΝΡα, ΙΡΝγ); cells producing at least 1b-2 and another cytokine (CE40b, ΤΝΡα, ΙΡΝγ); cells producing at least ΙΡΝγ and another cytokine (1b-2, ΤΝΡα, CE40b); cells producing at least ΤΝΡα and another cytokine (1b-2, CE40b, ΙΡΝγ).

An enhanced CE4 T-cell immune response is considered such a response when the number of cells producing any of the aforementioned cytokines is higher after administration of the adjuvant composition compared to the administration of the non-adjuvant composition. Typically, at least one, preferably two of the five conditions mentioned above will be fulfilled. In a particular embodiment, cells producing all four cytokines will be present in a larger amount in the adjuvant group compared to the non-adjuvant group.

Enhanced CE4 T cell immune response due to the adjuvanted anti-influenza composition of the present invention, ideally, can be obtained after a single injection. A single dose approach will be extremely suitable, for example, in a rapidly developing epidemic situation. In some cases, especially for the elderly, or in the case of young children (under the age of 9) who are vaccinated for the first time against influenza, or in the case of pandemics, it may be useful to administer 2 doses of the same composition during the season. The second dose of the same composition (also considered as the composition for the first vaccination) can be administered during the primary immune response and after an adequate period of time. Typically, a second dose of such a composition is given after a few weeks or about 1 month, for example 2, 3, 4, 5 or 6 weeks after the first dose, to help prioritize the immune system of non-responding or weakly responding individuals.

In a specific embodiment, administering said immunogenic composition alternatively or additionally induces an enhanced B-cell secondary immune response in patients who have been given an adjuvant immunogenic composition compared to a B-cell secondary immune response induced in individuals immunized with a non-adjuvant composition. An enhanced B-cell secondary immune response is understood to mean an increased incidence of peripheral blood B-lymphocytes capable of differentiating into antibody-secreting plasma cells after encountering an antigen, as measured by stimulating differentiation of ίη νίίτο (see sections of examples, for example, EuroRanalysis ( Ephuts-Npksb pptipoyugsSh

- 10 011419 κροΐ) B-cell memory (secondary immune response)).

In yet another specific embodiment, vaccination with the first adjuvant vaccination composition does not have a measurable effect on the SI8 response.

The inventors unexpectedly found that a composition containing an influenza virus or antigenic preparation made with an adjuvant in the form of an oil-in-water emulsion, in particular with an adjuvant in the form of an oil-in-water emulsion containing a metabolizable oil, sterol, such like alpha-tocopherol, and an emulsifier, is effective in stimulating T-cell responses in a weakened immune population. As shown by the inventors, the administration of a single dose of the immunogenic composition for the first vaccination, as described in this invention, can provide improved seroprotection, as assessed by the correlates of protection for influenza vaccines after revaccination of the elderly population against influenza compared with vaccination without adjuvant antiviral vaccine. The claimed adjuvant composition is also capable of inducing an enhanced SI4 T cell immune response against influenza virus compared with the response obtained using a non-adjuvant composition. This observation may be associated with increased reactivity after vaccination or infection with respect to antigenic stimulation by influenza virus. In addition, this may also be due to cross-reactivity, i.e. higher ability to respond to variant strains of influenza virus. Such an enhanced response may be especially useful for a human population with weakened immunity, such as the elderly population (aged 65 years and older), and in particular the elderly population from the high-risk group. This can lead to a decrease in the coefficient of general morbidity and mortality and the prevention of emergency hospitalization in the case of pneumonia and other flu-like diseases. It may also be beneficial for children (less than 5 years old, preferably less than 2 years old). In addition, this allows the induction of a CE4 T cell response, which is more stable over time, for example, is still present 1 year after the first vaccination, compared with the response induced by the adjuvant composition.

Preferably, the CE4 T cell immune response, such as an enhanced CE4 T cell immune response obtained from an unreceived subject, includes the induction of a cross-reactive CE4 T helper response. In particular, the number of cross-reactive CE4 T cells is increasing. By a cross-reactive CE4 response is meant recognition by CE4 by T cells of common epitopes among influenza virus strains.

The commonly available influenza vaccines are effective only against infectious influenza virus strains having hemagglutinins with similar antigenic characteristics. If the infectious (circulating) influenza virus has undergone minor changes (such as a point mutation or the accumulation of point mutations leading to amino acid changes, for example, in surface glycoproteins, in particular hemagglutinin (an antigenic drifting version of the virus strain)), then the vaccine can still provide some protection, although it can provide only limited protection, since newly created variants may elude immunity induced by a previous influenza virus infection or aktsinatsiey. Antigenic drift is responsible for the annual epidemics that occur during periods of time between pandemics (^ 11eu & 8x11s1. 1987, App. Isu. Wuset. 56, 365-394). The induction of cross-reactive SI4 T cells provides an additional advantage for the composition of the invention in that it can also provide cross protection, in other words, protection against heterologous infections, i.e. infections caused by a circulating strain of influenza virus, which is an option ( for example, drifting) strain of influenza virus contained in the immunogenic composition. This may be advantageous when a circulating strain is difficult to cultivate on chicken embryos or to produce in tissue culture, which makes the use of a drifting strain a working alternative. This may also be advantageous in cases where the subject has received the first and second vaccinations at intervals of several months or a year and when the influenza virus strain in the immunogenic composition used for the second immunization is a drifting variant strain of the strain used in the composition used for the first vaccination.

Therefore, the adjuvanted anti-influenza immunogenic composition, as defined herein, has a higher ability to induce serological protection and cross-reactive SI4 T cells in vaccinated elderly subjects. This property may be associated with a greater ability to give an answer against the variant strain of the strain that is presented in the immunogenic composition. This can serve as evidence of an important advantage in a pandemic situation. For example, a polyvalent anti-influenza composition containing any strain or several strains of H5, H2, H9, H7 or H6 can provide a greater ability to respond against a pandemic option, i.e. a drifting strain of said pandemic strain (s), both after the vaccination following this drifting strain, and after infection with it.

Detection of cross-reactive SI4 T cells after vaccination with influenza vaccine

After the introduction of the classic trivalent influenza vaccine (3 weeks) is observed

- 11 011419 a significant increase in the frequency of CE4 occurrence of peripheral blood T cells that respond to the preparation of an antigenic strain (whole virion or cleaved antigenic) that is homologous to one of the vaccines present (Η3Ν2: A / Rapata / 2007/99, Η1Ν1: Λ / Νο \ ν Ca1ebop1a / 20/99, B: B / 8yapdbopd / 7/97) (see Example III). A comparable increase in the frequency of occurrence can be observed upon repeated stimulation of CE4 T cells of peripheral blood with influenza virus strains classified as drifting strains (Η3Ν2: A / 8ubpeu / 5/97, Η1Ν1: A / Wetschd / 262/95, B: B / Uatapa51i / 166/98).

Conversely, if CE4 peripheral blood T cells are re-stimulated with influenza virus strains classified by expert in this field as shift strains (Η2Ν2: A / 8shdarote / 1/57, Η9Ν2: Λ / Ηoidkoid / 1073/99), no noticeable no increase after vaccination is observed.

CE4 T cells that are capable of recognizing both homologous and drifting strains of the influenza virus are referred to herein as cross-reactive. Adjuvant anti-influenza compositions, as set forth herein, are capable of demonstrating heterosubtypic cross-reactivity, since there is a noticeable cross-reactivity against drifting strains of the influenza virus. As indicated above, the ability of a pandemic vaccine composition to be effective against drifting pandemic strains can be considered an important characteristic in the case of pandemics.

According to the above observations in humans, CE4 T-cell epitopes were identified that are common for different strains of the influenza virus (Ce1beg C. e! A1., 1998, 1n1. 1tipo1. 10 (2): 211-22; CeEg S.M. e! A1 ., 1996, I. Uyo1., 70 (7): 4787-90; SeIet S.M. e! A1., 1995, I. Uyo1., 1995, 69 (12): 7497-506).

In a specific embodiment, the adjuvant composition may be of additional benefit, providing better protection against circulating strains that have undergone a more significant change (such as genetic recombination, for example, between two different species) in hemagglutinin (antigenic shift) against which currently not available vaccines effective. Other adjuvants

The composition may contain an additional adjuvant, in particular an adjuvant such as TKT-4 ligand, preferably a non-toxic derivative of lipid A. A suitable TKT-4 ligand is 3de-O-acylated monophosphoryl lipid A (ZP-MPB). Other suitable TKB-4 ligands are lipopolysaccharide (bP8) and derivatives, MEP (muramyl dipeptide) and protein P from P8U (respiratory syncytial virus).

In one embodiment, the composition may further include a ligand of a To11-like receptor (TKK-4) 4, such as a non-toxic derivative of lipid A, in particular monophosphoryl lipid A, or, more specifically, 3-de-O-acylated monophosphoryl lipid A ( ZP-MRI).

30-MPB is sold under the trade name MPB® (in this specification MPB) by Sopha sogrotiop and stimulates mainly CE4 + T-cell responses with the ΙΡΝ-γ phenotype (TM). It can be obtained according to the methods disclosed in CB 2220211 A. From a chemical point of view, it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Preferably, small particles of 30-MBP are used in the compositions of the present invention. Small 30-MPB particles have such a particle size that they can be sterile filtered through a 0.22 μm filter. Such production methods are described in \ UO 94/21292 and in example II.

30-MRB can be used, for example, in an amount of 1-100 μg (w / v) per dose of the composition, preferably in an amount of 10-50 μg (w / v) per dose of the composition. A suitable amount of 30-MPB is, for example, any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49 or 50 μg (w / v) per dose of the composition. More preferably, the amount of 30-MPB varies from 25 to 75 μg (w / v) per dose of the composition. Typically, the dose of the composition will range from about 0.5 to about 1 ml. A typical vaccine dose is 0.5, 0.6, 0.7, 0.8, 0.9, or 1 ml. In a preferred embodiment, a final concentration of 50 μg of 30-MBP is contained in 1 ml of the vaccine composition or 25 μg in 0.5 ml of the vaccine dose. In other preferred embodiments, a final concentration of 35.7 or 71.4 μg of 30-MPB is contained in 1 ml of the vaccine composition. Specifically, a 0.5 ml vaccine dose volume contains 25 or 50 μg of 30-MRP per dose.

The dose of MPI, respectively, is able to enhance the immune response to the antigen in humans. In particular, a suitable amount of MPI is one that improves the immunological potential of such a composition as compared to a non-adjuvant composition or as compared to a composition supplemented with a different amount of MPI adjuvant, while remaining acceptable in terms of reactogenicity profile.

Synthetic derivatives of lipid A are known, some of which are described as TBR-4 agonists and include, but are not limited to:

OM174 (2-deoxy-6-o- [2-deoxy-2 - [(K.) - 3-dodecanoyloxytetradecanoylamino] -4-o-phosphono-b-P-glucopyranosyl] -2 - [(K) -3- hydroxytetradecanoylamino] -a-O-glucopyranosylhydrogen phosphate) (\ UO 95/14026);

OM 294 ER (38.9K.) - 3 - [(K.) - dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (B) - [(B) -3-hydroxytetradecanoylamino] decan-1,10-diol , 1,10-bis (dihydrogen phosphate) (\ UO 99/64301 and \ UO 00/0462);

OM197 MR-As ER (38.9 K.) - 3 - [(K.) - dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 - [(K.) - 3-guide

- 12 011419 roxytetradecanoylamino] decane-1,10-diol, 1-dihydrogen phosphate, 10- (6-aminohexanoate) (νθ 01/46127).

Other suitable TKB-4 ligands are, for example, lipopolysaccharide and its derivatives, muramyl dipeptide (ΜΌΡ) or respiratory syncytial virus protein P.

Another suitable immunostimulant for use in the present invention is Οιιί1 A and its derivatives. Osh1A is a saponin preparation isolated from the South American tree Osh11a) a caroiapa toNpa. and was first described by Iakdaagb et al. in 1974. (8aroish abschuayk, Agsuyu. £ ig Fs decatel UlSh8 £ og8syyid, Uo1. 44, 8ppppdeg Ueg1ad, Veg1sh, p. 243-254) as having adjuvant activity. Purified fragments of Οιιί1 A were isolated by B ^ C, which retains the adjuvant activity without toxicity associated with Οιιί1 A (ΕΡ 0362278), for example 087 and 0821 (also known as 0A7 and 0A21). 08-21 is a naturally occurring saponin isolated from 0sh11a bark) and caroiapa toyia, which induces ί``8 + cytotoxic T cells (CTb), YOU cells and the predominant 1dC2antensive response, and is the preferred saponin in the context of the present invention.

Particular compositions 0821, which are particularly preferred, are described; these compositions further comprise sterol (νθ 96/33739). The saponins forming part of the present invention may be in the form of an oil-in-water emulsion (νθ 95/17210).

Revaccination and composition used for revaccination (composition for booster immunization)

According to an aspect of the present invention, there is provided the use of an influenza virus antigen in the manufacture of an anti-influenza immunogenic composition for revaccinating people pre-vaccinated with the polyvalent anti-influenza composition of this disclosure or said polyvalent anti-influenza composition containing a variant strain of influenza virus made with an adjuvant type oil in the form of an adjuvant oil in water as defined herein.

Typically, revaccination is carried out at least 6 months after the first vaccination (s), preferably after 8-14 months, more preferably after about 10-12 months.

An immunogenic composition for revaccination (a composition for booster immunization) may contain any type of antigenic preparation, either inactivated or lively attenuated. It may contain the same type of antigenic preparation, i.e. split influenza virus or antigenic preparation of this split flu virus, whole virion, purified HA and NA (subunit vaccine) or virosome, as well as the immunogenic composition that was used for the first vaccination. Alternatively, the booster immunization composition may contain a different type of influenza antigen, i.e. split influenza virus or antigenic preparation of this split flu virus, whole virion, purified HA and NA (subunit vaccine) or virosome, and not the one used for the first vaccination. A split viral or whole-virion vaccine is preferably used. The booster immunization composition may be adjuvant or non-adjuvant. The non-adjuvant booster immunization composition may be P1iapx ™ / a-K1x® / 1pLi8r1y®, which is administered intramuscularly. This composition contains three inactivated cleaved virion antigens prepared from WHO recommended influenza virus strains of the corresponding season.

Accordingly, a preferred embodiment of the invention provides the use of influenza virus or its antigenic preparation in the manufacture of an immunogenic composition for revaccinating people previously vaccinated with the immunogenic composition that is claimed in this specification.

The booster immunization composition may be adjuvant or non-adjuvant. In a preferred embodiment, the booster immunization composition comprises an oil-in-water emulsion adjuvant, in particular an oil-in-water emulsion adjuvant containing a metabolizable oil, a sterol, such as alpha-tocopherol, and an emulsifier. Preferably, said adjuvant in the form of an oil-in-water emulsion preferably contains at least one metabolizable oil in an amount of from 0.5 to 20% of the total volume and has oil droplets whose diameters are at least 70% in intensity are less than 1 micron.

In a preferred embodiment, the first vaccination is carried out with an influenza composition, preferably a split influenza virus composition containing at least one influenza virus strain that could potentially cause an outbreak of a pandemic, and a booster vaccination is carried out with an influenza composition containing at least one strain that is a circulating pandemic strain.

In a specific embodiment, the immunogenic revaccination composition (also referred to herein as the booster immunization composition below) comprises an influenza virus or antigenic preparation thereof that has common CE4 T cell epitopes with the influenza virus or antigenic preparation used for the first vaccination. A common CE4 T cell epitope is considered to mean peptides / sequences / epitopes from different antigens that can be recognized by the same CO4 cell (see examples of the described epitopes in Ce1beg C. e1 a1., 1998, 1S. 1ttio1. 10 (2): 21122; CeEg S.M. e1 a1., 1996, 1. U1go1. 70 (7): 4787-90; CeEg S.M. e1 a1., 1995, 1. Upo1. 69 (12): 7497-506).

In an embodiment according to the invention, the booster immunization composition is a monovalent influenza composition containing a strain of influenza virus that is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. Suitable strains are

- 13 011419 by themselves Η5Ν1, Η9Ν2, Η7Ν7, Η2Ν2 and Η1Ν1, but are not limited to this. The specified strain may be the same or one of those that are present in the composition used for the first vaccination. In an alternative embodiment, said strain may be a variant strain, i.e. a drifting strain of that strain that is present in the composition used for the first vaccination.

In another specific embodiment, the booster immunization composition is a multivalent influenza vaccine. In particular, when the booster immunization composition is a multivalent influenza vaccine, such as a divalent, trivalent or tetravalent vaccine, at least one strain is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. In a specific embodiment, two or more strains in the booster immunization composition are pandemic strains. In another specific embodiment, at least one pandemic strain in the booster immunization composition is a strain of the same type or one that is present in the composition used for the first vaccination. In an alternative embodiment, at least one strain may be a variant strain, i.e. a drifting strain of at least one pandemic strain that is present in the composition used for the first vaccination.

Accordingly, in another aspect of the present invention, there is provided the use of an influenza virus or an antigenic preparation thereof from a first pandemic strain of an influenza virus in the manufacture of an immunogenic composition for protecting against influenza caused by an influenza virus strain that is a variant of said first influenza virus strain.

Accordingly, in another aspect of the present invention, the use of:

(a) an influenza virus or antigenic preparation thereof from a first influenza virus strain; and (b) an oil-in-water emulsion adjuvant, as defined herein, in the manufacture of an immunogenic composition for protection against influenza caused by an influenza virus strain which is a variant of the indicated first strain of influenza virus.

The booster immunization composition may be adjuvant or non-adjuvant.

Typically, a booster immunization composition, if used, is used for the next flu season, for example about 1 year after the first immunogenic composition. The composition for booster immunization can also be used every subsequent year (third, fourth, fifth vaccination, etc.). The booster immunization composition may be the same as the composition used for the first vaccination. Accordingly, the booster immunization composition comprises an influenza virus or antigenic preparation thereof, which is a variant of the influenza virus strain used for the first vaccination. In particular, influenza virus strains or their antigenic preparation are selected according to a reference substance distributed by the World Health Organization, so that they are adapted to the influenza virus strain circulating in the year of revaccination.

The influenza virus antigen or antigenic composition used for revaccination preferably contains an adjuvant or an oil-in-water emulsion, respectively, as described above. The adjuvant may be an oil-in-water emulsion adjuvant, as described hereinabove, which is preferred, optionally containing an additional adjuvant, such as a TBB-4 ligand, such as, for example, 3Ό-ΜΡΕ or saponin, or may be another suitable adjuvant, such as alum or alternatives to alum, such as polyphosphazene.

Preferably, when revaccination induces any one, preferably two or all, of the following: (1) an enhanced CE4 response against the influenza virus or its antigenic drug, or (2) an enhanced B-cell secondary immune response, or (3) an enhanced humoral response compared with the equivalent response induced after the first vaccination using a non-adjuvant influenza virus or its antigenic preparation. Preferably, when the immunological responses induced after revaccination with the adjuvanted influenza virus or its antigenic preparation, as defined herein, are higher than the corresponding response induced after revaccination with the non-adjuvant composition. Preferably, the immunological responses induced after revaccination using a non-adjuvant, preferably cleaved, influenza virus are higher in a population of a first time vaccinated adjuvant composition of a preferentially cleared influenza virus, compared to the corresponding response in a population, a first time vaccinated with a non-adjuvant influenza virus composition, preferably a cleaved virus .

As demonstrated by the inventors, revaccination of subjects with a booster immunization composition containing an influenza virus and an oil-in-water emulsion adjuvant containing a metabolizable oil, a sterol, such as alpha-tocopherol, and an emulsifier, as defined hereinabove, show more high antibody titers compared with the corresponding values in the group of people who were first vaccinated with the adjuvant composition and re-stimulated with the adjuvant composition. The effect of the adjuvant on enhancing the antibody response to revaccination is especially important for the elderly, who are known to have a low response to vaccination or infection with influenza virus. Success associated with adjuvant compositions has also been noted with respect to enhancing the CE4 T-cell response after revaccination.

The adjuvant composition of the invention is capable of inducing a better cross-reactant

- 14 011419 anti-drift strain (strain of influenza virus from the next influenza season) compared with the protection provided by the control vaccine. The indicated cross-reactivity showed a longer persistence compared to the reactivity obtained using the adjuvanted vaccine. The effect of the adjuvant on enhancing cross-reactivity against the drifting strain is important for a pandemic situation.

The preclinical data shown, for example, in Example 3, demonstrate the ability of the composition of the invention to protect against infection with a heterotypic influenza virus and against influenza, as measured by body temperature recording. The same conclusion holds true for the results of clinical trials obtained in revaccination studies.

In another embodiment, the present invention relates to a vaccination regimen according to which the first vaccination is carried out by a composition of an influenza virus, preferably a composition of a cleaved influenza virus containing at least one strain of influenza virus that could potentially cause an outbreak of a pandemic, and revaccination is carried out by a circulating strain, or pandemic strain, or classic strain.

SE4 epitope in NA

This antigenic drift is mainly characteristic of the epitope regions of the viral surface proteins of hemagglutinin (HA) and neuraminidase (ΝΑ). It is known that any difference in CE4- and B-cell epitopes among different strains of the influenza virus used by the virus to escape from the adaptive response of the host immune system will play an important role in vaccination against influenza, and this does occur.

CE4 T-cell epitopes common to different strains of the influenza virus have been identified in humans (see, for example, Ce1eeg C. e! A1., 1998, Ιηί. 1tipo1. 10 (2): 211-22; Ce1eeg S.M. e ! a1., 1996, 1. νίτοί. 70 (7): 4787-90; and SeIet S.M. e! a1., 1995, 1. νίτο1., 69 (12): 7497-506).

In a specific embodiment, booster vaccination is carried out by using a booster immunization composition comprising an influenza virus or antigenic preparation thereof that has common CE4 cell epitopes with an influenza virus antigen or antigenic preparation used for the first vaccination. Thus, the present invention relates to the use of an immunogenic composition comprising a pandemic influenza virus or antigenic preparation thereof and an oil-inlet emulsion adjuvant, in particular an oil-in-water emulsion adjuvant containing a metabolizable oil, a sterol, such as alpha-tocopherol, and an emulsifier, in the manufacture of a multi-dose vaccine component for a first vaccination, the multi-dose vaccine further comprising, in the form of a repeated immunizing dose, an influenza virus or an antigenic preparation thereof, a cat ing a common SE4 T-cell epitopes with an antigen of pandemic influenza virus or antigenic preparation of the virus present in a dose, administration of the first vaccination.

Vaccination products

The composition of the invention can be administered by any suitable means of delivery, such as intradermal, via mucous membranes, for example, intranasal, oral, intramuscular or subcutaneous. Other delivery methods are well known in the art.

For an adjuvanted influenza composition, an intramuscular delivery method is preferred.

Intradermal delivery is another suitable method. For intradermal delivery, any suitable device can be used, for example, devices with a short needle, such as the devices described in I8 4886499, I8 5190521, I8 5328483, I8 5527288, I8 4270537, I8 5015235, I8 5141496, I8 5417662. Intradermal vaccines can also be administered by devices that limit the effective length of the needle penetration into the skin, such as the devices described in νθ 99/34850 and EP 1092444, which are incorporated herein by reference, and their functional equivalents. In addition, needleless injection devices are suitable for delivering liquid vaccines to the dermis by means of a needleless fluid injector or by means of a needle piercing the corneal layer and creating a stream reaching the dermis. Needleless injection devices are described, for example, in I8 5480381, I8 5599302, I8 5334144, I8 5993412, I8 5649912, I8 5569189, I8 5704911, I8 5383851, I8 5893397, I8 5466220, I8 5339163, I8 53 633 643, I8 5339163, 38 , I8 5520639, I8 4596556, I8 4790824, I8 4941880, I8 4940460, νθ 97/37705 and XVO 97/13537. In addition, ballistic powder / particle delivery devices that use compressed gas to accelerate the passage of the vaccine in powder form through the outer layers of the skin into the dermis are suitable. In addition, conventional syringes can be used in the classic intradermal mantoux route.

Another suitable route of administration is the subcutaneous route. Any suitable device may be used for subcutaneous delivery, such as a classic needle. Preferably use the action of a needleless injector, such as published in νθ 01/05453, νθ 01/05452, νθ 01/05451, νθ 01/32243, νθ 01/41840, νθ 01/41839, νθ 01/47585, νθ 01/56637, νθ 01/58512, νθ 01/64269, νθ 01/78810, νθ 01/91835, νθ 01/97884, νθ 02/09796, νθ 02/34317. More preferably, said device is pre-filled with a liquid vaccine composition.

- 15 011419

Alternatively, the vaccine is administered intranasally. Typically, a vaccine is administered topically to the nasopharyngeal region, preferably without pulmonary nebulization. It is desirable to use a device for intranasal delivery, which delivers the vaccine composition to the nasopharyngeal region, without or essentially without its penetration into the lungs.

Preferred devices for intranasal administration of the vaccines of the invention are nebulization devices. Suitable commercially available nasal spray devices include Assikrgau ™ (VesDop Oyktkop). Nebulizers produce a very fine aerosol that can be easily inhaled into the lungs, and therefore it will not reach the mucous membranes of the nose effectively. Therefore, nebulizers are not preferred.

Preferred nebulization devices for intranasal use are devices whose effectiveness is independent of the pressure applied by the user. These devices are known as threshold pressure devices. The liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve aerosol production with a regular droplet size. Threshold pressure devices suitable for use in the present invention are known in the art and are described, for example, in AO 91/13281, and EP 311863 B, and EP 516636, which are incorporated herein by reference. Such devices are commercially available from the company RDsDdeg Otn and are also described in Vottsg, J. PärtassiDsa1 Tssbnogo Eigors, 8crd. 1999.

Preferred intranasal devices produce droplets (measured using water as the liquid phase) in the range of 1-200 μm, preferably 10-120 μm. Below 10 microns there is a risk of inhaling them, therefore, it is desirable to have no more than about 5% droplets of less than 10 microns. Droplets larger than 120 μm do not spray as well as smaller droplets, so it is desirable to have no more than about 5% droplets in excess of 120 μm.

Double dose delivery is an additional preferred feature of the intranasal delivery system for use with the vaccines of the invention. Double dosing devices contain 2 sub-doses of a single vaccine dose, 1 sub-dose for administration to each nostril. Typically, 2 sub-doses are located in one compartment, and the design of the device allows efficient delivery of 1 sub-dose at a given time. Alternatively, a monodosing device may be used to administer the vaccine of the invention.

Alternatively, an epidermal or transdermal vaccination method is also included in the present invention.

In a specific aspect of the present invention, an adjuvant immunogenic composition for a first administration may be administered intramuscularly, and a booster immunization composition, either adjuvant or not, may be administered by another method, for example, intradermal, subcutaneous or intranasal. In another specific embodiment, the composition for the first administration may contain a standard amount of HA of 15 μg per strain of influenza virus, and the booster composition may contain a low dose of HA, i.e. less than 15 μg, and depending on the route of administration, may be administered in a smaller volume.

Vaccination populations

The target population for vaccination may be an immunocompromised person. People with weakened immune systems are usually less able to respond to an antigen, in particular an influenza virus antigen, compared to healthy adults.

Preferably, the target population is a population that is not primed against influenza, or that is naive (for example, in relation to a pandemic strain), or has not previously responded to an infection or vaccination with influenza virus. Preferably, when the target population is older people, respectively, at least 50, usually at least 55, or at least 60, or at least 65 years of age or older, younger high-risk adults (i.e. i.e., between the ages of 18 and 64), such as, for example, people working in health facilities or young adults with a risk factor such as cardiovascular and pulmonary disease or diabetes. Another target population is all children 6 months of age and older, especially children 6-23 months of age, who show a relatively high flu-related hospitalization rate. Preferably, the target population is older people over the age of 65.

Modes, dosage and additional criteria for the effectiveness of vaccination

Preferably, the immunogenic compositions of the present invention are a standard 0.5 ml injection dose, and in most cases it contains 15 μg of the hemagglutinin antigen component of each strain of the influenza virus, as measured by simple radial immunodiffusion (8NF) (EM. Aob cd a1., E Vu1. 8 Dapb. 5 (1977) 237-247; EM.Aoob sD a1., E Vyu1. 8Dapb. 9 (1981) 317-330). Preferably, the volume of the vaccine dose will be from 0.5 to 1 ml, in particular the standard volume of the vaccine dose of 0.5 or 0.7 ml. A small adjustment of the dose volume can be carried out in a standard way depending on the concentration of HA in the initial bulk sample.

Preferably, said immunogenic composition comprises a low dose of HA antigen, for example

- 16 011419 any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 μg of HA per strain of influenza virus. A suitable low dose of HA is from 1 to 7.5 μg of HA per strain of influenza virus, respectively, from 3.5 to 5 μg, for example 3.75 μg of HA per strain of influenza virus, usually about 5 μg of HA per strain of influenza virus.

Preferably, the vaccine dose of the invention, in particular the low vaccine dose, can be presented in a smaller volume than traditional injectable split-yi vaccines, which is usually about 0.5, 0.7 or 1 ml per dose. The small doses of the invention are preferably below 500 μl, more preferably below 300 μl, and most preferably not more than about 200 μl or less per dose.

Thus, a preferred small volume vaccine dose in accordance with one aspect of the invention is a dose containing a low dose of antigen in a small volume, for example about 15 μg, or about 7.5 μg HA, or about 3.0 μg HA (per strain) in a volume of approximately 200 μl.

The influenza drug of the invention preferably meets the specific international criteria for vaccines.

International standards have been adopted to determine the effectiveness of influenza vaccines. The official European Union (EU) criteria for an effective flu vaccine are given in the table below. 1. Theoretically, in order to meet the requirements of the European Union, an influenza vaccine should satisfy only one of the criteria listed in the table for all influenza virus strains included in the vaccine. The compositions of the present invention, respectively, satisfy at least one of these criteria.

However, in practice, it will be necessary that all strains satisfy at least two or all three criteria, in particular with regard to a new vaccine, such as a new vaccine for delivery by another method. In some cases, two criteria may suffice, for example, it may be acceptable for two of the three criteria to satisfy all strains, while the third criterion will satisfy several, but not all, strains (for example, two of the three strains). The requirements for the adult population (18-60 years old) and the elderly population (over 60 years old) are different.

Table 1

18-60 years old Over 60 years old Seroconversion Level * > 40% > 30% Conversion Factor ** > 2.5 > 2.0 Protection Level *** > 70% > 60%

* For each vaccine strain, the level of seroconversion is defined as the percentage of vaccinees who have at least a four-fold increase in serum titers of hemagglutinin (III) inhibition after vaccination.

** For each vaccine strain, the conversion factor is defined as a multiple increase in serum geometric mean titers (CMT) ΗΣ after vaccination.

*** The level of protection is defined as the percentage of those vaccinated with a serum W-titer> 1:40 after vaccination (for each vaccine strain), which is usually accepted as an indicator of protection.

In a further aspect, the invention provides a method for creating a vaccine against diseases that are known to be treated by CO4 + T cell activation, comprising:

1) the choice of antigen containing CO2 + epitopes, and

2) combining said antigen with an adjuvant in the form of an oil-in-water emulsion as defined herein above, wherein said vaccine, after administration to said mammal, is capable of inducing an enhanced CO4 T cell response in said mammal.

The contents of all references in this application, including patent applications and granted patents, are incorporated into this description in its entirety by reference.

In order to avoid ambiguity, the inventors assume that the terms containing, contain and contain in this description can be replaced in each case, respectively, by the terms consisting of, consist of and consists of.

The invention will now be described with reference to the following non-limiting examples.

Example I describes the immunological reading methods used in studies on mice, ferrets and humans.

Example II describes the preparation and characterization of an oil-in-water emulsion and adjuvant compositions used in the exemplary studies.

Example III describes a clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus and A803 adjuvant in an elderly population over the age of 65.

Example IV describes a second clinical trial (revaccination test) of a vaccine containing an antigenic preparation of a split influenza virus and A803 adjuvant in an elderly population

- 17 011419 over the age of 65 years.

Example Y shows a preclinical evaluation of adjuvant and non-adjuvant ferret influenza vaccines (study Ι and study ΙΙ). Carried out temperature monitoring, measured viral shedding and CE4 T-cell response.

Example U1 shows a preclinical evaluation of adjuvant and non-adjuvant influenza vaccines in naive and primed C57B1 / 6 mice.

Example VII shows a preclinical evaluation of adjuvant and non-adjuvant anti-influenza split and subunit vaccines in C57B1 / 6 mice primed with heterologous strains.

Example VIII describes a clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus containing adjuvant A803, adjuvant A803 + MPB or not containing any exogenous adjuvant in an elderly population over the age of 65 years.

Example IX shows the preclinical evaluation of adjuvant and non-adjuvant ferret influenza vaccines (study III). Carried out temperature monitoring, measured viral shedding and W-titers.

Example X shows a clinical trial of a vaccine containing an antigenic preparation of a split influenza virus containing A803 adjuvant with or without MPI in an elderly population over the age of 65: data on immunogenicity persistence on the 90th and 180th days.

Example XI shows a clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus containing adjuvant A803 with MPI in an elderly population over the age of 65 years.

Example XII shows a clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus containing adjuvant A803 with MPI in two concentrations in an elderly population over the age of 65 years.

Example I. Methods of immunological reading.

11. Methods on mice.

D1.1. Hemagglutination inhibition test.

Testing methodology

The titers of antihemagglutinin antibodies to three strains of the influenza virus were determined using a hemagglutination inhibition test (III). The principle of the W-test is based on the ability of specific anti-influenza antibodies to inhibit the hemogglutination of red blood cells (BBC) of the chicken with hemagglutinin (HA) of the influenza virus. Heat-inactivated serum samples were pretreated with kaolin and chicken air force to remove non-specific inhibitors. After pretreatment, two-fold dilutions of serum were incubated with 4 units. hemagglutination of each strain of influenza virus. Then chicken red blood cells were added and inhibition of agglutination was evaluated. The titers were expressed as the reciprocal of the highest dilution of serum at which complete inhibition of hemagglutination was observed. Since the first dilution of serum was 1:20, an undetectable level was evaluated as a titer of 10.

Statistical analysis

Statistical analysis was performed for W-titers after vaccination using IM8TAT. The protocol attached to the analysis of variance can be briefly described as follows:

logarithmic data conversion;

Shapiro-Wilk criterion (81κιρίΐΌ- \ νί11 <) for each population (group) in order to check the normal distribution of groups;

Cochren's criterion (СосЬпап) in order to check the dispersion uniformity between different populations (groups);

two-way analysis of variance performed on groups;

Tukey's ΗδΌ-test (Tiksu) for multiple comparisons (ск Й Й к к щ ш са Ш б | | |

D1.2. Staining of intracellular cytokines.

This technique allows the quantification of antigen-specific T lymphocytes based on their cytokine production: effector T cells and / or effector memory T cells produce ΣΕΝ-γ and / or central (central hematopoietic organs) memory T cells produce HB 2. On the 7th day after immunization, PBMCs (peripheral blood mononuclear cells) are collected.

Lymphoid cells re-stimulate w ν 11th in the presence of a secretion inhibitor (brefeldin). These cells are then subjected to the traditional immunofluorescence procedure using fluorescent antibodies (to CE4, CE8, γ-γ and B-2). The results are expressed as the frequency of occurrence of cytokine-positive cells among ΟΌ4 / ί.Ό8 T cells. Intracellular staining of T-cell cytokines was performed on PBMCs 7 days after the second immunization. Blood samples were taken from mice and pooled in BPMI heparinized medium (Rock \\ 's11 Ragk Mstop1 ShkShiU + Abb (additives). For blood, suspensions of PBB (peripheral blood lymphocytes) diluted with BPMNAbb were layered according to the Lutbo1y! C-Matt gradient recommended protocol (centrifugation

- 18 011419 min at 2500 rpm and room temperature). Mononuclear cells were removed from the phase boundary, washed 2x in KPM1 + Ay and PBMC suspensions were adjusted to 2x 10 ⁶ cells / ml using ΚΡΜΙ with 5% fetal calf serum (PC8).

Antigenic stimulation of ίη νίίτο PBMC was performed at a final concentration of 1x10 ⁷ cells / ml (PAC8 tube with whole ΡΙ (\ νΐιο1ο ΡΙ) (1 μg HA / strain) and then incubated for 2 hours at 37 ° C with the addition of anti-CE28 and anti-CE49 (1 μg / ml for both).

After the repeated antigenic stimulation step, PBMCs were incubated overnight at 37 ° C in the presence of brefeldin (1 μg / ml) at 37 ° C to inhibit cytokine secretion.

Staining on ΙΡΝ-γ / ΙΕ-2 / ί.Ό4 / ί.Ό8 was carried out as follows.

Cell suspensions were washed, resuspended in 50 μl of PB8 with 1% PC8 (phosphate buffered saline) containing 2% PC-blocking reagent (1/50; 2.462). After 10 min incubation at 4 ° C, 50 μl of a mixture of anti-CE4-PE (2/50) and anti-CE8 regSp (3/50) was added and incubated for 30 min at 4 ° C. After washing in PB8 with 1% PC8, the cells were permeabilized by resuspension in 200 μl of SuYuPx-SuYuregt (BO kit) and incubated for 20 min at 4 ° C. The cells were then washed using Regt \ ν; · ΐ51ι (BO kit) and resuspended using 50 μl of a mixture of anti-ΙΡΝ-γ APC (1/50) + anti-1E-2 P1TC (fluorescein isothiocyanate) (1/50), divorced in Regt \ ν; · ΐ51ι. After incubation for a minimum of 2 hours, a maximum of overnight at 4 ° C, the cells were washed with Regt; ·51ι and resuspended in PB8 with 1% PC8 + 1% paraformaldehyde. The analysis of the samples was carried out using PAC8 (Pioge5sepsse-asNta1 ce11 5g1eg cell sorter with activation of fluorescence). Living cells were sorted (P8C / 88C) and data were collected on approximately 20,000 events (lymphocytes) or 35,000 events on CE4 + T cells. ΙΡΝ-γ + or Ш2 + percentages were calculated on the sorted populations of CE4 + and SE8 +.

Ι. 2. Ferret methods.

Ι.2.1. Hemagglutination Inhibition Test (ΗΙ).

Testing methodology

The titers of antihemagglutinin antibodies to three strains of the influenza virus were determined using a hemagglutination inhibition test (ΗΙ). The principle of the теста test is based on the ability of specific anti-influenza antibodies to inhibit hemogglutination of chicken erythrocytes (CVC) by hemagglutinin (HA) of the influenza virus. Serum samples were initially treated with a 25% solution of neuraminidase (ΚΌΕ) and heat inactivated to remove non-specific inhibitors. After pretreatment, two-fold dilutions of serum were incubated with 4 units. hemagglutination of each strain of influenza virus. Then chicken red blood cells were added and inhibition of agglutination was evaluated. The titers were expressed as the reciprocal of the highest dilution of serum at which complete inhibition of hemagglutination was observed. Since the first dilution of serum was 1:10, an undetectable level was evaluated as a titer of 5.

Statistical analysis

Statistical analysis was performed for ΗΙ-titers (41 days before the challenge) using υΝΙ8ΤΛΤ. The protocol attached for analysis of variance can be briefly described as follows:

logarithmic data conversion;

Shapiro-Wilk test (81ι; ψίΓ0- \ νί11 <) for each population (group) in order to check the normality of the distribution in groups;

Cochren's criterion (СосЬпап) in order to check the dispersion uniformity between different populations (groups);

interaction criterion for one-way ANVA (analysis of variance);

Tukey's ΗδΌ-test for multiple comparisons.

Ι.2.2. Body temperature monitoring.

Individual temperatures were recorded during the challenge infection period using sensors and telemetry recordings. All implants were checked and updated, and before placement in the intraperitoneal cavity, a new calibration was carried out using Ό8а (Ea1a 8aepse§ ENetAnopae Seiaigioted 123, 5015 TS Tyssd, Netherlands). At the time of these measurements, all animals were individually housed in separate cages. Temperatures were recorded every 15 min for 4 days before the challenge and up to 7 days after the challenge.

Ι.2.3. Nasal swabs.

Nasal swabs were obtained by introducing 5 ml of PB8 into both nostrils of awake animals. The inoculum was collected in a Petri dish and placed in sample containers on dry ice.

Titration of viruses in nasal washes

All nasal samples were first filtered under sterile conditions through 8px X filters (CoMag) to remove any bacterial contamination. 50 μl of serial ten-fold dilutions of nasal washings were transferred to microtiter plates containing 50 μl of medium (10 wells / dilution). Then, 100 μl of MOSCOW cells (2.4 × ^{10 5} cells / ml) were added to each well and incubated

- 19 011419 whether at 35 ° C for 5-7 days.

After 5-7 days of incubation, the culture medium is carefully removed, 100 μl of 1/20 U8T-1-containing medium are added and incubated for another 18 hours.

The intensity of the yellow formazan dye resulting from the reduction of U8T-1 (water-soluble tetrazolium salt, from English \\ 'a (ex 5o1i1e1e1ga5o1shtai) viable cells is proportional to the number of viable cells in the well at the end of the analysis of virus titration and is determined quantitatively by measuring the titration of viruses and is determined quantitatively absorbance in each well at the corresponding wavelength (450 nm). The reference point is defined as the average value of ΘΌ (optical density) for uninfected control cells is 0.3 ΘΌ (0.3 ΘΌ corresponds to +/- 3 8Ό (standard deviation) ΘΌ for uninfected control cells.) A positive score is determined when ΘΌ is below the reference point, and conversely, a negative score is determined when ΘΌ is above the reference point. Viral shedding titers were determined according to Kei aiy Mieisy and expressed as 1od of TSS ₅ ,. (Average cytopathogenic dose (infecting 50% of cells)) / ml.

Σ.3. Assays for assessing the immune response in humans.

Σ.3.1, Analysis of hemagglutination inhibition.

The immune response was determined by measuring W-antibodies using the method described by the WHO Center for the Joint Study of Influenza (LUNO Sopaogayid Seikge Σογ Shlie ^ a), Centers for Disease Control, Atlanta, USA (1991).

Measurement of antibody titer was performed on thawed serum samples using a standardized and comprehensively reliable micromethod using 4 units. hemagglutination inhibition (4 NGOs) of the corresponding antigens and a 0.5% suspension of chicken red blood cells. Nonspecific serum inhibitors were removed by heat treatment and treatment with a receptor-degrading enzyme.

The resulting serum samples were evaluated by levels of W-antibodies. Starting from the initial dilution of 1:10, a series of dilutions (2 times) was prepared until the final dilution, inclusive, of 1: 20480. The highest dilution was taken as the titration endpoint, at which complete inhibition (100%) of hemagglutination was observed. All analyzes were performed in duplicate.

D3.2. Neuraminidase Inhibition Assay.

The analysis was carried out in fetuin-coated microtiter plates. A series of double dilutions of antiserum was prepared and mixed with a standardized amount of influenza A virus No. N2, H1M or influenza B. The test is based on the biological activity of neuraminidase, which enzymatically releases neuraminic acid from fetuin. After cleavage of the terminal neuraminic acid, β-E-galactose-M-acetylgalactosamine becomes available. Horseradish peroxidase (NKR) -labeled agglutinin from peanut Agasic yurodaea, which specifically binds to galactose structures, was added to the wells. The amount of agglutinin bound can be determined and quantified by reaction with tetra-methylbenzidine (TMB) peroxidase substrate. The highest dilution of antibodies, which still inhibits the activity of viral neuraminidase by at least 50%, was indicated as M-titer.

P3.3. Analysis of neutralizing antibodies.

Measurements of neutralizing antibodies were performed on thawed serum samples. The neutralization of the virus contained in serum antibodies was determined in the analysis of microneutralization. Serums were used in this assay without further processing. Each serum was tested in triplicate. A standardized amount of virus was mixed with serial dilutions of the serum and incubated to allow the antibodies to bind to the virus. Then, a cell suspension containing a certain number of ISKCON cells was added to the mixture of virus and antiserum and incubated at 33 ° C. At the end of the incubation period, virus replication was visualized using chicken erythrocyte hemagglutination. A serum titer corresponding to 50% neutralization was calculated by the method of Kei and Mieisy.

P3.4. Cell-mediated immunity was assessed using flow cytometry cytokines (CPO).

Antigen-specific ΟΌ4 and ΟΌ8 peripheral blood T cells can be re-stimulated 1 and νίίτο to produce (P-2, СО40,,-alpha and ΛΝ, if incubated with their corresponding antigen. As a result, the amount of antigen-specific ΟΌ4 and ΟΌ8 T -cells can be counted by flow cytometry using traditional immunofluorescence labeling of the cell phenotype, as well as the intracellular production of cytokines. In this study, to re-stimulate influenza-specific T cells using whether the influenza vaccine virus antigen, as well as peptides of a particular influenza virus protein, were expressed as the frequency of occurrence of cytokine-positive ΟΌ4 or в8 T cells in a ΟΌ4 or ΟΌ8 T cell subpopulation.

P3.5. Statistical methods.

D3.5.1. Primary endpoints.

Percentage, intensity, and association with local and general signs and symptoms of vaccination during the 7-day follow-up period (i.e., on the day of vaccination and for the next 6 days) after vaccination and in general.

- 20 011419

The percentage, intensity, and association with vaccination of local and general signs and symptoms of non-anxiety during the 21-day follow-up period (i.e., on the day of vaccination and for the next 20 days) after vaccination and in general.

Cases of serious adverse events throughout the study.

Ι.3.5.2. Secondary endpoints.

Regarding the humoral immune response: observed variables:

on days 0 and 21: titers of serum hemagglutination-inhibiting (ΗΙ) antibodies and ΝΙ antibodies, individually tested against each of the three influenza virus strains presented in the vaccine (anti-HCH1-, anti-H3№- and anti B antibodies);

on days 0 and 21: titers of neutralizing antibodies, tested individually against each of the three strains of the influenza virus presented in the vaccine;

secondary variables (with 95% confidence intervals):

geometric mean titers (SMT) of serum антител antibodies with 95% confidence intervals (95% CI) before and after vaccination;

seroconversion levels * from 95% CI on day 21;

conversion factors ** from 95% CI on day 21;

seroprotection levels *** with 95% CI on day 21;

SMT serum ΝΙ antibodies (with 95% confidence intervals) for all time points.

* Seroconversion level, defined as the percentage of vaccinees who have at least a four-fold increase in serum тит titers on day 21 compared to day 0 for each vaccine strain.

** Conversion factor, defined as a multiple increase in serum CMT ΗΙ on day 21 compared with day 0, for each vaccine strain.

*** The level of protection, defined as the percentage of vaccinees with a serum тит-titer of 40, after vaccination (for each vaccine strain), which is usually taken as an indicator of protection.

As for the cell-mediated immune (SMC response:

observed variable:

on days 0 and 21: the frequency of occurrence of cytokine-positive CO4 / CO8 cells by 10 ⁶ in different tests. In each test, the response of CO4 / CO8 T cells to the peptide antigen of influenza virus (ρί) was quantified (the exact nature and origin of these antigens needs to be presented / explained);

split influenza virus antigen (§ £);

whole influenza virus antigen (\\ T);

secondary variables:

cells producing at least two different cytokines (CE40E, ΙΕ-2, ΙΕΝγ, ΕΝΕα);

cells producing at least CO40b and another cytokine (ΙΕ-2, ΊΝΕα, ΙΕΝγ);

cells producing at least ΙΕ-2 and another cytokine (CO40, ΊΝΕα, ΙΕΝγ); cells producing at least ΙΕΝγ and another cytokine (ΙΕ-2, ΊΝΕα, СО40Б);

cells producing at least TIR and another cytokine (ΙΕ-2, СО40Б. ΙΕΝγ).

Ι.3.5.3. Immunogenicity analysis.

An immunogenicity assay was performed for the entire cohort of vaccinees. For each treatment group, the following parameters were calculated (at 95% confidence intervals):

geometric mean titers (SMT) of ΗΙ- and ΝΙ-antibodies on days 0 and 21;

geometric mean titers (SMT) of neutralizing antibodies on days 0 and 21;

conversion factors on the 21st day;

seroconversion levels (8C) on day 21, defined as the percentage of vaccinees who have at least a four-fold increase in serum тит-titers on day 21 compared to day 0;

protection levels on the 21st day, defined as the percentage of vaccinees with serum titre equal to 1:40;

the frequency of occurrence of CO4 / CO8 T-lymphocytes secreting in response was summarized (descriptive statistics) for each vaccinated group, for each time point (day 0, day 21) and for each antigen (influenza virus peptide (ρί), split influenza virus (T) and whole influenza virus (\\ T));

descriptive statistics of the individual differences between responses at time points after and before (Ρθδΐ-Рge) for each vaccinated group and each antigen (ρί, ίи \\ Т) in each of 5 different tests;

to compare the differences found between the 3 groups, a nonparametric criterion was used (the Kruskal-Wallis test (Kgi5ka11- ^ aSh5)) and the statistical value p was calculated for each antigen in each of 5 different tests. All criteria of significance were bilateral. P values of less than or equal to 0.05 were considered statistically significant.

- 21 011419

Example II Preparation and characterization of an oil-in-water emulsion and adjuvant compositions.

Unless otherwise indicated, the oil-in-water emulsion used in the following examples consists of an organic phase prepared from two types of oils (alpha-tocopherol and squalene) and an aqueous phase of ΡΒ8. containing tween 80 as an emulsifier. Unless otherwise indicated, the oil-in-water emulsion adjuvant compositions used in the following examples were prepared as containing the following oil-in-water emulsion components (final concentrations are given): 2.5% squalene (v / v) vol.), 2.5% alpha-tocopherol (vol / vol), 0.9% polyoxyethylene sorbitan monooleate (vol / vol) (Tween 80), see νΟ 95/17210. This emulsion, referred to in the following examples as L803, was prepared as a double concentrate.

ΙΙ.1. Preparation of the emulsion 8Β62.

ΙΙ.1.1. Laboratory-scale cooking.

Tween 80 is dissolved in phosphate buffered saline (ΡΒ8) to give a 2% solution in ΡΒ8. To obtain 100 ml of a two-fold concentrate of the emulsion, 5 g of OB-alpha-tocopherol and 5 ml of squalene are intensively mixed for complete mixing. Add 90 ml of ΡΒδ / Tween solution and mix to mix completely. The resulting emulsion is then passed through a syringe and the final microfluidization is carried out using the MyugoiShsk M1108 apparatus. The resulting oil droplets have a size of approximately 120-180 nm (expressed as average Ζ, measured by ΡC8). Other adjuvants / antigenic components are added to the emulsion by simple mixing.

ΙΙ.1.2. Cooking on a larger scale.

The preparation of emulsions 8–62 is carried out by mixing, under conditions of strong mixing, the oil phase consisting of hydrophobic components (α-tocopherol and squalene) and the aqueous phase containing water-soluble components (Tween 80 and ΡΒδ-mode (modified); pH 6.8) . With stirring, the oil phase (1/10 of the total volume) is transferred to the aqueous phase (9/10 of the total volume) and the mixture is stirred for 15 minutes at room temperature. The resulting mixture is then subjected to shear, shock and cavitation forces in the microfluidizer interaction chamber (8 cycles of 15,000 psi (103.4 MPa)) to produce submicron droplets (distribution from 100 to 200 nm). The resulting pH value is 6.8 ± 0.1. The emulsion 8–62 is then sterilized by filtration through a 0.22 μm membrane and the sterile bulk emulsion is stored in a refrigerator in Siras containers at 2–8 ° C. The dead volume of a container with a final bulk emulsion of 8–62 is purged with sterile inert gas (nitrogen or argon) for at least 15 seconds.

The final composition of the emulsion 8Β62:

Tween 80: 1.8% (v / v) 19.4 mg / ml; squalene: 5% (v / v) 42.8 mg / ml; α-tocopherol: 5% (v / v) 47.5 mg / ml; ΡΒδ-mode: No. 101 121 mm, KS1 2.38 mm, No. ₂ ΗΡΟ ₄ 7.14 mm, ΚΗ ₂ ΡΟ ₄ 1.3 mm; pH 6.8 ± 0.1.

ΙΙ. 2. Oil droplet size measurement using dynamic light scattering.

ΙΙ.2.1. Introduction

The diameter of the oil droplets is determined according to the following procedure and in the following experimental conditions. Measurement of droplet size is given as a measure of intensity and is expressed as the average ζ measured by ΡC8.

ΙΙ.2.2. Sample preparation.

Size measurements were carried out for the adjuvant in the form of an oil-in-water emulsion: 8–62, prepared according to the enlarged scale preparation method, L803 and Ά803 + ΜΡΕ (50 μg / ml), the last two adjuvants were prepared immediately before use. The composition of the samples is given below (see section ΙΙ.2.4). Samples were diluted 4000x-8000x at ΡΒ8 7.4.

For control, particle standards of Ρ ^ -Naηosa1 with a size of 100 nm (catalog number 6011-1015) were diluted in 10 mM No. C1.

ΙΙ.2.3. The results of the measurement of dimensions on the Layout

All size measurements were performed using both Layout Ζеίа8 ^ ζе ^ 3000 ^8.

Samples were measured in a plastic cuvette for Makeg analysis in a suitable dilution (usually with dilutions from 4000x to 20,000x depending on the concentration of the samples) and using two optical models:

either the actual refractive index (ΚΙ) of the particle is 0 and the imaginary is 0, or the real refractive index of the particle is 1.5 and the imaginary is 0.01 (adapted optical model for the emulsion according to the values found in the literature).

The following specifications were installed:

laser wavelength: 532 nm (Geΐa3000Η8), laser power: 50 mW ^ Ιπ3000Η8), scattered light detected at 90 ° (Ζеΐа3000Η8), temperature: 25 ° С, duration: programmed automated determination, quantity: 3 consecutive measurements,

- 22 011419 average diameter ζ: according to cumulative analysis, size distribution: according to the SopP method or the method using an automated algorithm.

The automated Layout algorithm uses a combination of the algorithm for cumulative analysis, the SOPP algorithm, and the non-negative least squares algorithm (ΝΝΣ8).

The intensity distribution can be converted into a volume distribution thanks to the M1e theory. ΙΙ.2.4. Results (see table. 2).

Cumulant analysis (average diameter Ζ)

table 2

Sample Breeding Record Count rate ΖΑϋ Polydispersity ZV62 5000 one 7987 153 0.06 2 7520 153 0.06 3 6586 152 0,07 average 7364 153 0.06 8V62 8000 one 8640 151 0,03 (Example IV) 2 8656 151 0.00 3 8634 150 0.00 average 8643 151 0.01 8B62 + MP1_ 25 8000 one 8720 154 0,03 mcg (*) 2 8659 151 0,03 3 8710 152 0.02 average 8697 152 0.02

(*) Prepared as follows: water for injection, PB8 10x concentrated, 250 μl of 8B62 emulsion and 25 μg of ICBMs are mixed together with a final volume of 280 μl.

The size of the average diameter ζ (ΖΑΏ) is estimated by the amount of light scattered by particles of each size in the sample. This value is associated with monomodal analysis of the sample and is used mainly for reproducibility purposes.

Count rate (SC) is a measure of scattered light: it corresponds to thousands of photons per second.

The polydispersity index (Po1y) is the width of the distribution. It is a dimensionless criterion for broadening the distribution.

Analysis by Soibp or using an automated algorithm

Two other preparations 8B62 (double concentrated L803) were prepared and evaluated according to the procedure explained above, with the following minor modifications.

Samples were measured in a plastic cuvette for Makegp analysis in two dilutions to obtain optimal count rates: 10000x and 20000x for Ge8a8 ^ ze ^ 3000N8, in the same optical models as used in the above example. The results are shown in table. 3.

Table 3

ΙΚ

Sipe analysis (average in nm)

8V62

1022

1/10000 breeding

1/20000

1023

1/10000

1/20000

Valid

1,5 0

1,5

1,5 0

1,5

Intensity 149

158

159 161 158 161 154

160

Volume

167

139

200

141

198

140

185

133

Analysis by automaton, algorithm (average Intensity

150

155

155

147 '

155

150

151

154 in nm) Volume

143

196

144

182

- When the obtained values were incoherent.

A schematic representation of these results is shown in FIG. 1 for composition 1023. As can be seen, the vast majority of particles (eg, at least 80%) have a diameter of less than 300 nm in intensity.

ΙΙ.2.5. General conclusion.

Composition 8B62 was measured at different dilutions using the Layout SΖa8 ^ ζе ^ 3000Н8 and two optical models. The particle size ΖΆΌ (i.e., the average value of the intensity according to the cumulative analysis) of these compositions, evaluated above, was approximately 150-155 nm.

When using the cumulative algorithm, the inventors did not observe any effect of dilution on ΖΑΏ and polydispersity.

- 23 011419

ΙΙ. 3. Preparation A803. containing MRI.

ΙΙ.3.1. Preparation of a liquid suspension of MPI.

Liquid bulk MPB (as used throughout this document, is an abbreviated version for 3P-MPB, i.e. 3-O-deacylated monophosphoryl lipid A), is prepared from lyophilized MPB® powder. Liquid bulk MPI is a stable concentrated (approximately 1 mg / ml) aqueous dispersion of a crude material that is ready for use in a vaccine or adjuvant composition. A schematic representation of the cooking method is shown in FIG. 2.

For a maximum batch size of 12 g, the preparation of liquid bulk MPI is carried out in sterile glass containers. The MPI dispersion process consists of the following stages:

suspend MPI powder in water for injection;

disaggregate any large aggregates by heating (heat treatment); reduce particle size to a range from 100 to 200 nm by microfluidization;

carry out preliminary filtration of the drug on the module for pre-filtering 8ag1oc1eap. 0.8 / 0.65 microns;

sterile filtration of the drug at room temperature (module SAG1O2Gap P; 0.22 μm).

The MPB powder is lyophilized by microfluidization to obtain a stable colloidal aqueous dispersion (MPB particle size less than 200 nm). Lyophilized MPI powder is dispersed in water for injection to obtain a coarse suspension of 10 mg / ml. Then the suspension is subjected to heat treatment with stirring. After cooling to room temperature, the microfluidization process is started to reduce particle size. Microfluidization is performed using the installation M1sgoDi161S5 M110EN, by continuously circulating the dispersion through the chamber for mikroflyuidizatsionnyh interactions at a certain pressure for a minimum amount of passages (number of cycles: n _{m; n).} The microfluidization duration, represented by the number of cycles, is calculated based on the measured flow rate and the dispersion volume. On this equipment at the specified pressure, the resulting flow rate can vary from one interaction chamber to another and over the life of a particular interaction chamber. In the presented example, the interaction chamber used is of the type Ε20Υ M1sgoDi161C5. Since microfluidization efficiency is associated with a pressure-flow rate pair, the duration of the process can vary from one batch to another. The time required for 1 cycle is calculated based on the flow rate. It should be borne in mind that the flow rate is the flow rate measured using water for injection immediately before the introduction of MPI in the installation. One cycle is defined as the time (in minutes) required for one passage of the total volume of MPI through the installation. The time required to obtain n cycles is calculated as follows:

the number of MPI for processing (ml) / flow rate (ml / min)

Thus, in accordance with this select the number of cycles. The minimum number of execution cycles ( _pf ) is described for the preferred equipment and interaction chambers used. The total number of work cycles is determined by the result of particle size measurements performed after _five cycles. Based on the literature data, the limiting particle size is determined (6 11 _t ). The measurement is performed by a technique of photon correlation spectroscopy (RS8) and b _1ip expressed as unimodal result (2 _ategade). If a value is obtained below this limit, then microfluidization can be stopped after five _PT cycles. Above this limit, microfluidization is continued until a satisfactory reduction in size is obtained, for a maximum of 50 subsequent cycles.

If filtration is not carried out immediately after microfluidization, then the dispersed MPB is stored at a temperature of +2 to + 8 ° C, awaiting transmission to the filtration area.

After microfluidization, the dispersion is diluted with water for injection and sterilely filtered through a 0.22 μm filter in a laminar flow. The final concentration of MPI is 1 mg / ml (0.80-1.20 mg / ml).

ΙΙ.3.2. Preparation of A803 + MPE-adjuvant vaccine: approach in 1 bottle.

MPA was added to the A803 adjuvant composition at a final concentration of 10-50 μg per vaccine dose.

Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as a mixture of 8B62 containing tween, Triton X-100 and UE8 (vitamin E succinate), are added to water for injection. The quantities of detergents are taken taking into account the detergents present in the strains of the influenza virus in order to achieve the target final concentration of 750 μg / ml Tween 80, 110 μg / ml Triton X-100 and 100 μg / ml UE8. After 5 minutes of stirring, 15 μg of each strain of influenza virus of interest is added (e.g., strain Η1Ν1, Η3Ν2 and B in a classic trivalent vaccine). After 15 minutes of stirring, 250 μl of 8B62 emulsion is added and then 25 μg or 50 μg MPB.

A schematic representation of the cooking method is shown in FIG. 3. The final composition A803, containing MPI, per dose for humans is shown in table. 4.

- 24 011419

Table 4

Ingredients Concentration Per dose for humans Title Component number Other 8V62 Squalene (solution 43 mg / ml) Tocopherol (solution 48 mg / ml) Tween 80 (solution 20 mg / ml) 781 μl / ml 250 μl 10.68 mg 11.86 mg 4.85 mg MP1 _ ** (solution 1 mg / ml) 78 mcg / ml or 156 mcg / ml 25 mcg or 50 mcg RVZ-mod * No. C1 KS1 Ma ₂ NRA ₄ KN ₂ RO ₄ 137 mm 2.7 mm 8.1 mm 1.47 mm 2.56 mg 0.064 mg 0.368 mg 0.064 mg Water for injections Add. 320 μl pH 6.8 ± 0.1

* PB8-mode tenfold concentrated, pH 6.8 KN ₂ PO ₄ , No. ₂ 1 ΙΡΟ ₄ , NaCl, KC1-HC1. ** MRI is either 25 μg or 50 μg per dose.

ΙΙ.3.3. Preparation of L803 + MPB adjuvant vaccine: approach in 2 vials.

The same composition can be prepared using the approach in 2 vials by mixing twice concentrated antigen or antigenic preparation with the adjuvant L803 (250 μl 8B62) or L803 + MPB (250 μl 8B62 + 25 μg or 50 μg MPB). In this case, the preparation is as follows. The preparation of the L825-adjuvanted influenza vaccine consists of three main stages:

1) preparation of the trivalent final bulk composition (double concentrated) without adjuvant and placement in a container for antigens;

2) preparation of adjuvant L803 + MPI;

3) dilution immediately before use of L803 + MPB-adjuvant cleaved viral vaccine.

1) Preparation of the trivalent final bulk composition without adjuvant and placement in an antigen container.

Volumes of three monovalent bulk forms are taken based on the HA content measured in each monovalent bulk form before preparation of the composition and a target volume of 1100 ml. A concentrated, phosphate-buffered saline solution and a pre-prepared mixture of Tween 80, Triton X-100 and α-tocopheryl hydrosuccinate are diluted in water for injection. Then, three concentrated bulk monoforms (АЖе \ at Sa1ebosh, ЛЖе \ at Wogk, W / Iapji) are successively diluted in the resulting solution, which is a phosphate buffered saline / Tween 80 - Triton X-100 - α-tocopheryl hydrosuccinate (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8.1 mM LHRO4, 1.47 mM KH2RO4, 990 μg / ml Tween 80, 150 μg / ml Triton X-100 and 130 μg / ml α-tocopheryl hydrosuccinate), to obtain the final concentration of 39.47 μg of HA from strains A (NSh1, ΙΙ3Ν2) in 1 ml of the trivalent final bulk form (15 μg of HA / strain A / 380 μl of the trivalent end bulk form) and 46 μg of HA from strain B (17.5 μg of HA / strain B / 380 μl of the trivalent final bulk form). Between the addition of each monovalent bulk form, the mixture is stirred for 10-30 minutes at room temperature. After the addition of the last monovalent bulk form and 15-30 minutes of stirring, the pH is monitored and adjusted to 7.2 ± 0.2 using HC1 or No. O11.

The trivalent final bulk form of antigens is placed under aseptic conditions in 3 ml sterile glass vials of type I (European Pharmacopoeia). Each vial has a volume of 470 μl (380 μl + 90 μl considering overflow).

2) Preparation of the bulk form of adjuvant A803 / MPB and placement in a container for adjuvants.

Adjuvant A803 / MPB is prepared by mixing two components: emulsion 8B62 (method in section A.1.2) and MPB (method in section A.3.1). A single concentrated PB8 mode (prepared by diluting a tenfold concentrated PB8 mode in water for injection) is mixed with bulk form 8B62 and liquid bulk form MPB at a concentration of 1 mg / ml. The concentration of MPI will be determined in such a way as to achieve a final content in the range of 10-50 μg, respectively, approximately 25 μg per final vaccine dose for humans. The mixture is stirred for 5-30 minutes at room temperature and the pH is adjusted to 6.8 ± 0.1 using No. O11 (0.05 or 0.5 M) / HC1 (0.03 M or 0.3 M). After further stirring for 5-30 minutes at room temperature, the mixture was sterilized by filtration through a 0.22 μm membrane. For at least 1 min, purge with sterile inert gas (nitrogen) to create a free inert space in the filled containers. A803 + MPB sterile adjuvant is stored at + 2-8 ° C until aseptic filling in 1.25 ml sterile glass syringes of type Ι (European Pharmacopoeia). Each syringe has an excess volume of 80 μl (320 μl + 80 μl considering overflow).

- 25 011419

At the time of injection, the contents of a pre-filled syringe containing adjuvant are injected into a vial that contains concentrated trivalent inactivated cleaved virion antigens. After mixing, the contents are returned back to the syringe, and the needle is replaced with a needle for intramuscular injection. 1 dose of reconstituted L5> 25-adjuvanted influenza vaccine candidate corresponds to 0.7 ml.

ΙΙ. 4. Preparation of immunogenic compositions containing an influenza virus antigen and possibly MPI in an oil-in-water emulsion composition.

An equal volume of twofold concentrated antigen of the cleaved influenza virus (P1iapx ™) (15 μg HA per strain) was added to the 8B62 emulsion from S.1. And mixed. This was combined, when appropriate, with 50 μg / ml MPB to give the final composition.

Example ΙΙΙ. A clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus and A803 adjuvant in an elderly population over the age of 65 (Exp1o-P1i-001).

Phase I of an open, randomized trial was performed in an elderly population over the age of 65 in 2003. in order to assess the reactogenicity and immunogenicity of the influenza vaccine-candidate of 61ao8tyyK1che (68K) Vysokosha containing adjuvant L803. The humoral immune response (i.e., titers of anti-hemagglutinin, neutralizing and antineuraminidase antibodies) and a cell-mediated immune response (CE4 and / or CE8 T cell responses) were measured 21 days after intramuscular administration of 1 dose of the L803 adjuvant vaccine or AUU vaccine. Iiaph ™ was used for comparison.

ΙΙΙ.1. Study plan.

Three groups of subjects who simultaneously received the following vaccine intramuscularly:

one group of 50 subjects receiving 1 dose of reconstituted and adjuvanted 8U influenza vaccine (P1iL803);

one group of 50 subjects receiving 1 dose of the whole-virion vaccine (P1iAUU); one group of 50 subjects receiving 1 dose of P1iaph ™ (P1iaph), which is a control group.

Vaccination schedule: 1 injection of influenza vaccine on day 0, blood sampling, data analysis on day 21 (determination of ΗΙ antibodies, determination of антител antibodies, determination of neutralizing antibodies and analysis ΟΜΙ) and the conclusion of the study.

The standard trivalent split P1iaph ™ influenza vaccine used in this study is commercially available since 2003. A vaccine developed and manufactured by 61Aho8tyyK1she Vyuoshchosh.

ΙΙΙ. 2. Vaccine composition and administration (table. 5).

ΙΙΙ.2.1. Vaccine preparation

L803-Adjuvant influenza vaccine

The L803 Adjuvant Influenza Vaccine Candidate is a 2-component vaccine consisting of concentrated trivalent inactivated cleaved virion antigens presented in an Ι type glass vial (335 μl) (antigen container) and a Ι type prefilled glass syringe containing 8B62 emulsion (335 μl) (container for adjuvants). At the time of injection, the contents of the antigen container are removed from the vial using a syringe pre-filled with 8B62 emulsion, followed by gentle mixing of the contents of the syringe. Mixing 8B62 emulsion with vaccine antigens gives the L803 adjuvant. Before injection, the used needle is replaced with an intramuscular injection needle and the volume is adjusted to 500 μl.

the dose of reconstituted L803-adjuvanted influenza vaccine corresponds to 0.5 ml, contains 15 μg of HA of each strain of influenza virus, as in the registered R1iapkh ™ / a-K1x® vaccine, and contains 10.68 mg of squalene, 11.86 mg of E-alpha tocopherol and 4.85 mg of polysorbate 80 (tween 80).

Cooking

The preparation of the L803-adjuvanted influenza vaccine consists of three main stages.

1) Preparation of the trivalent final bulk composition without adjuvant and placement in an antigen container.

The volumes of the three monovalent bulk forms are taken based on the HA content measured in each monovalent bulk form before preparation of the composition and the target volume of 800 ml. A concentrated, phosphate-buffered saline solution and a pre-prepared mixture of Tween 80, Triton X-100 and α-tocopheryl hydrosuccinate are diluted in water for injection. Then, three concentrated bulk monoforms (strain Α / No. \ ν Ca1ebop1a, strain A / Rapata and strain B / 8yapdbopd) are successively diluted in the resulting solution, which is phosphate buffered saline / Tween 80 - Triton X-100 - α-tocopheryl hydrosuccinate (pH 7.4, 137 mM No. C1, 2.7 mM KCl, 8.1 mM Νη ₂ ΗΡΟ4, 1.47 mM ΚΗ ₂ ΡΟ ₄ , 1500 μg / ml Tween 80, 220 μg / ml Triton X-100 and 200 μg / ml α-tocopheryl hydrosuccinate), to obtain a final concentration of 60 μg of HA from strains A in 1 ml of the trivalent final bulk form (15 μg of HA / strain A / 250 μl of three alentnoy final bulk form) and 70 micrograms HA from strain B (17.5 ug HA / strain B / 250 l trivalent final bulk form). Between the addition of each monovalent bulk form, the mixture is mixed.

- 26 011419 sew for 10 min at room temperature. After the addition of the last monovalent bulk form and 15 minutes of stirring, the pH is monitored and adjusted to 7.2 ± 0.2 with HC1 or NaOH.

The final trivalent bulk form of antigens is placed under aseptic conditions in 3 ml Type I sterile glass vials. Each vial has an excess volume of 34% (total volume 335 μl).

2) Preparation of 8B62 sterile bulk emulsion and placement in an adjuvant container.

Water phase: with stirring, 902 ml of Tween 80 are mixed with 44105 ml of PB8-mod buffer (pH is 6.8 after summing with HC1).

Oil phase: 2550 ml of squalene are added to 2550 ml of α-tocopherol with stirring.

Mixing of the aqueous and oil phases: with stirring, 5000 ml of the oil phase (1/10 of the total volume) is transferred to 45007 ml of the aqueous phase (9/10 of the total volume). The mixture was stirred for 15 minutes at room temperature.

Emulsification: the resulting mixture is subjected to shear, shock and cavitation forces in the microfluidizer interaction chamber (8 cycles of 15,000 psi (103.4 MPa)) to obtain submicron droplets (distribution from 100 to 200 nm). The resulting pH value is in the range of 6.8 ± 0.1.

Sterile filtration: the 8B62 emulsion is sterilized by filtration through a 0.22 μm membrane and the sterile bulk emulsion is stored in a refrigerator in Siras containers at 2-8 ° C. The dead volume of the container with the final bulk emulsion 8B62 is purged with sterile inert gas (nitrogen or argon) for at least 15 seconds.

All amounts of ingredients are given for the preparation of 50 l of emulsion and are given in volumes. In practice, these quantities are weighed based on the density of the ingredients. The density of PB8 is considered to be 1. The final composition of the 8B62 emulsion is as follows.

Table 5

The 8B62 sterile bulk emulsion is then placed under aseptic conditions in 1.25 ml Type I sterile glass syringes. Each syringe has an excess volume of 34% (total volume 335 μl).

3) Dilution immediately before use of the A803 adjuvant cleaved viral vaccine.

At the time of injection, the contents of the vial containing concentrated trivalent inactivated cleaved virion antigens are removed from the vial using a syringe containing 8B62 emulsion, followed by gentle mixing of the contents of the syringe. Mixing 8B62 emulsion with vaccine antigens gives A803 adjuvant.

ΙΙΙ.2.2. The vaccine composition (table. 6) and the introduction.

Table 6

Vaccine Composition Group Niaph ™ HA from 3 strains of influenza virus (total HA = 45 μg): • Α / Νβνν Ca1ec1ota / 20/99 (ΐνΚ-116): 15 mcg • A / Rapata / 2007/99 (ΚΕδνΐΚ-17): 15 mcg • B / 8Lapds1opd / 7/97: 15 mcg Thiomersal Content: 5 mcg In 0.5 ml pre-filled syringes Piaph νννν HA from 3 strains of influenza virus (total HA = 45 μg): • Α / Νβνν Ca1ec! Op1a / 20/99 (ΐνΚ-116): 15 mcg • A / Rapata / 2007/99 (KE8U1K-17): 15 mcg • B / 8Iapds1opd / 7/97: 15 mcg Thiomersal Content: 5 mcg In 0.5 ml vials E1iU \ AA / Niaph + A803 HA from 3 strains of influenza virus (total HA = 45 μg): • Α / Νβνν Ca1ebop1a / 20/99 (ΐνΚ-116): 15 mcg ' • A / Rapata / 2007/99 (ΡΕδνΐΚ-17): 15 mcg • B / 8Lapds1opd / 7/97: 15 mcg Thiomersal Content: 5 mcg In a bottle of 0.335 ml (2-fold concentrated) + in a syringe (0.335 ml) containing an 8B62 oil-in-water emulsion (preparation on an enlarged scale). Yai-A803

- 27 011419

Vaccines were injected intramuscularly into the deltoid muscle of the non-dominant arm. The vaccinees were carefully monitored for at least 30 minutes, with appropriate medical treatment being readily available in the event of an infrequent anaphylactic reaction after administration of the vaccine.

ΙΙΙ. 3. Results of population studies.

In this study, a total of 148 subjects were registered: 49 subjects in the P1iA803 group, 49 subjects in the P1iapkh group and 50 subjects in the P1iAUU group. The mean age of the entire cohort of vaccinees at the time of vaccination was 71.8 years with a standard deviation of 6.0 years. The average age of the subjects and their gender distribution among the three vaccine groups were similar.

ΙΙΙ. 4. Safety Conclusions.

The administration of the influenza vaccine with adjuvant A803 was safe and clinically well tolerated by the examined population, i.e. older people over the age of 65.

ΙΙΙ. 5. Immunogenicity results.

An immunogenicity assay was performed for the entire cohort of vaccinees.

ΙΙΙ.5.1. Humoral immune response.

In order to evaluate the humoral immune response induced by the A803 adjuvant vaccine, the following parameters were calculated for each treatment group (at 95% confidence intervals):

geometric mean titers (OMT) of ΗΙ- and ΝΙ-antibodies on days 0 and 21;

geometric mean titers (OMT) of neutralizing antibodies on days 0 and 21;

seroconversion levels (8C) on day 21, defined as the percentage of vaccinees who have at least a four-fold increase in serum тит-titers on day 21 compared to day 0;

conversion factors on day 21, defined as a multiple increase in serum OMT ΗΙ on day 21 compared to day 0, for each vaccine strain;

protection levels on day 21, defined as the percentage of vaccinees with a serum тит titer of 1:40.

ΙΙΙ.5.1.1. Antihemagglutinin antibody response.

a) Geometric mean titers (OMT) ΗΙ.

OMT for ΗΙ antibodies with 95% CI are shown in table. 7 (OMT for anti-S antibody). Before vaccination, OMT antibodies for all vaccine strains were in the same range for three groups. After vaccination, anti-hemagglutinin antibody levels increased significantly. After vaccination, there was a trend towards higher OMT ΗΙ antibody values for all three vaccine strains in the P1iA803 and P1iag1x groups, although there was some overlap of 95% CI between the P1iapx groups and the P1iAUU groups.

Table 7

Antibody Group Schedule N ΘΜΤ Value 95% CI ίί υί Α / Νβνν NiA803 Rge 49 25.6 17.3 37.9 Ca1ec1op1a Niaph ΡΙ (ϋ21) 49 317.7 219.1 460.7 P1i \ LLL / Rge 49 26.3 18.1 38,4 ΡΙ (ϋ21) 49 358.5 244.2 526.4 Rge fifty 19.7 13.6 28.6 ΡΙ (02) fifty 138.2 90.3 211.7 A / Rapata NIAZOZ Rge 49 52.3 35,4 77.4 Niaph ΡΙ (ϋ2) 49 366.1 264.5 506.6 None \ LLL / Rge 49 40.9 28.1 59.5 ΡΙ (021) 49 296.0 205,4 426.6 Rge fifty 25.8 18.0 37.1 ΡΙ (021) fifty 165.6 116.0 236.5 B / 8papds1opd NIAZOZ Rge 49 27.5 19.0 39.8 Niaph ΡΙ ϋ21) 49 317.7 226.9 444.9 NIUL / UU Rge 49 26.0 17,2 39.2 ΡΙ (021) 49 270,0 187.0 389.7 Rge fifty 32,0 20.8 49.3 ΡΙ (021) fifty 195.6 135.2 282.9

N = number of subjects with available results.

95% CI = 95% confidence interval; IU = lower limit; u = upper limit. MGMMAX = minimum / maximum.

RYE = before vaccination on day 0.

RCE21) = after vaccination on the 21st day.

b) Conversion factors for titers of anti-J-antibodies, seroprotection levels and seroconversion levels (correlate with protection in humans).

The results are presented in table. 8.

- 28 011419

Conversion factors represent a multiple increase in serum OMT ΗΙ for each vaccine strain on day 21 compared to day 0. The conversion factor varies from 6.1 to 13.6 depending on the viral strain and the vaccine. This conversion factor significantly exceeds the twofold increase in OMT required by European authorities.

Seroprotection levels represent the proportion of subjects with a serum тит titer greater than or equal to 40 on day 21. At the beginning of the study, half of the subjects (range 34.0-69.4%) in all groups had protective antibody levels for all strains. On the 21st day, seroprotection levels in three groups ranged from 88.0 to 100% for different viral strains. In the context of immunity, this means that more than 88% of subjects had a serum ΗΙ titer after vaccination greater than or equal to 40, and it is believed that they are protected against these three strains. This level significantly exceeds the level of seroprotection of 60%, required by European departments for the elderly population aged 60 years or older.

Seroconversion levels represent the proportion of subjects with at least a four-fold increase in serum тит titers on day 21 compared to day 0. The levels of general response for the three strains were essentially equal in the three groups. To be considered effective and consistent with the requirements of the European Union (EU), the vaccine must induce a seroconversion rate of more than 30% in the elderly population aged 60 years. In this study, seroconversion levels exceeded 50% for the three groups.

Table 8

Seroprotection Level Seroconversion rate Conversion factor EU Standard (> 60 years old) > 60% > 30% > 2.0 Strains Group N % [95% CI] % [95% CI] Media [95% CI] Α / Νβνν Sa1edogpa Yaiazoz 49 98.0 [89.1-99.9] 69.4 [54.6-81.7] 12.4 [7.3-21.0] Piaph 49 98.0 [89.1-99.9] 69.4 [54.6-81.7] 13.6 [8.0-23.2] Pi \ LLL / fifty 88.0 [75.7-95.5] 52.0 [37.4-66.3] 7.0 [4.0-12.2] A / Rapata Yaiazoz 49 100.0 [92.7- 100.0] 55.1 [40.2-69.3] 7.0 [4.2-11.6] Piaph 49 91.8 [80.4-97.7] 65.3 [50.4-78.3] 7.2 [4.7-11.3] Yai \ L / \ L / fifty 90.0 [78.2-96.7] 56.0 [41.3-70.0] 6.4 [3.9-10.4] IN/ 8 Lapds1opd Yaiazoz 49 100.0 [92.7- 100.0] 73.5 [58.9-85.1] 11.6 [7.2-18.6] Piaph 49 95.9 [86.0-99.5] 69.4 [54.6-81.7] 10.4 [6.5-16.5] Yai \ LLL / fifty 90.0 [78.2-96.7] 50.0 [35.5-64.5] 6.1 [3.6-10.3]

N = total number of subjects.

Finally:

after vaccination, there was a tendency to higher OMT ΗΙ antibody values for all three vaccine strains in the P1iA803 and P1iapch groups, although there was some overlap of 95% CI between the P1iapk groups and Ρ1πννν groups;

the conversion factor varies from 6.1 to 13.6 depending on the viral strain and the vaccine. Such a conversion factor significantly exceeds the twofold increase in OMT required by European departments;

on the 21st day, seroprotection levels in the three groups ranged from 88.0 to 100% for different viral strains. This level significantly exceeds the level of seroprotection of 60% required by European authorities for the elderly population aged 60 years or older;

in this study, seroconversion levels exceeded 50% for the three groups. The levels of general response for the three strains essentially turned out to be equal in the three groups.

ΙΙΙ.5.1.2. Antibody titers.

In order to better characterize the immune response to influenza vaccination in the elderly, serum antibody responses to neutralizing antigens were evaluated. The results are shown in table. 9 (seroprotection levels and geometric mean titers (OMT) for antineutral antibody titers) and table. 10 (seroconversion levels for antineutralization on the 21st day after vaccination (a multiple increase is 4)).

Antibody titers against three strains of influenza virus were measured in serum before and after immunization. The following parameters were determined:

geometric mean titers (OMT) of serum neutralizing antibodies with 95% confidence intervals (95% CI) before and after vaccination;

- 29 011419 seroconversion levels with 95% CI on day 21, defined as the percentage of vaccinees having at least a four-fold increase in W titers on day 21 compared to day 0 for each vaccine strain.

Table 9

Antibody Gr. Schedule N > = 18 1 / ϋΙΙ smt η % 95% CI Value 95% CI and υι B be Α / Νβνν one REE 49 46 93.9 83.1 98.7 106.6 77.6 146.6 Ca1ec1op1a ΡΙ (ϋ21) 49 49 100.0 92.7 100.0 870.2 608.5 1244.3 2 REE 49 48 98.0 89.1 99.9 115.6 89.4 149.5 ΡΙ (ϋ21) 49 49 100.0 92.7 100.0 955.8 649.5 1406.5 3 REE fifty 46 92.0 80.8 97.8 87.7 63.6 120.8 ΡΙ (ϋ21) fifty fifty 100.0 92.9 100.0 375.4 271.2 519.6 A / Rapata one REE 49 49 100.0 92.7 100.0 724.7 558.0 941.1 ΡΙ (ϋ21) 49 49 100.0 92.7 100.0 2012.8 1438.4 2816.5 2 REE 49 49 100.0 92.7 100.0 727.8 556.1 952.6 ΡΙ (ϋ21) 49 49 100.0 92.7 100.0 1597.7 1128.8 2261.5 3 REE fifty fifty 100.0 92.9 100.0 512.0 409.3 640.6 RC021) fifty fifty 100.0 92.9 100.0 977.8 738.2 1295.0 V / Ziapd- one REE 49 29th 59.2 44,2 73.0 25.6 18.8 35.0 s1opd ΡΙ (ϋ21) 49 48 98.0 89.1 99.9 222.5 148.1 334.2 2 REE 49 27 55.1 40,2 69.3 29.3 20.1 42.7 ΡΙ (ϋ21) 49 49 100.0 92.7 100.0 190.4 127.6 284.3 B / b 3 REE fifty 31 62.0 47.2 75.3 33,4 23.1 48,4 s1opd ΡΙ (ϋ21) fifty 46 92.0 80.8 97.8 117.8 82.6 168.0

Group 1: G1i vaccine = adjuvant and 2x conc. Ii woo.

Group 2: AI vaccine = None vaccine.

Group 3: None vaccine = None \ U'U'U vaccine.

N = number of subjects with available results.

p /% = number / percentage of subjects with a titer in a specific range.

95% CI = 95% confidence interval; B = lower limit; v = upper limit. RKE = before vaccination on day 0.

RCV21) = after vaccination on the 21st day.

Table 10

Group 3: NI-vaccine (HBi59A2) None HU'U'U'-vaccine.

N = number of subjects with available results both before and after vaccination.

n = number of responders.

% = share of responders (η / Νχ100).

95% CI = strictly 95% confidence interval; YU = lower limit, GU = upper limit.

Key Results

For three vaccines on the 21st day, a seroprotection level of 100% was obtained against both strains A. As for strain B, the seroprotection levels in the three groups ranged from 92 to 100%.

After vaccination, a significant increase in OMT was observed for all strains in the three groups. However, there was a tendency to higher OMT neutralizing antibody values for all three vaccine strains in the G1iA803 and G1iapkh groups compared to the G1i \ U’U'U 'group, although there was some overlap of 95% CI between the G1ialkh groups and the G1iUUU group.

As for the levels of seroconversion, the levels of general response for the three strains, essentially, were equal in the three groups.

In all groups, the results were consistent with the results obtained from the analysis performed for antihemagglutinin antibodies.

- 30 011419

S.5.1.3. Antibody titers against neuraminidase (ΝΆ).

In order to better characterize the immune response to influenza vaccination in the elderly, serum antibody responses to neuraminidase antigens were evaluated. By analogy with the titer of W-antibodies, the following endpoints were determined:

SMT (taking the antilogarithm from the average for the logarithmic transformation of the titles);

seroconversion level, defined as the percentage of vaccinees having at least a four-fold increase in W-titers on day 21 compared to day 0 for each vaccine strain.

SMT and seroconversion levels for W-antibodies with 95% CI are shown in table. 11 (SMT anti-NoA antibodies) and tab. 12 (NΆ seroconversion levels after vaccination (21st day) (fourfold increase)).

Table 11

Antibody Group Schedule N (ZMT 95% CI be Α / Νθνν Pyazosis RNE 49 77.8 61.8 97.9 Ca1ec1op1a ΡΙ (ϋ21) 48 270,0 212.9 342.3 Iiaph RNE 49 77.8 64.6 93.6 ΡΙ (ϋ21) 49 249.1 190.0 326.5 Ni'LL / RNE fifty 66.8 53.8 83.0 ΡΙ (ϋ21) fifty 159.2 122.8 206.4 A / Rapata Pyazosis RNE 49 33.3 28.5 48.7 ΡΙ (ϋ21) 48 156.8 124.8 196.9 Iiaph RNE 49 34.2 25.6 45.8 ΡΙ (ϋ21) 49 133.7 100.9 177.3 Pi \ LLL / RNE fifty 24.6 18.7 32,4 ΡΙ (ϋ21) 49 78.9 59,4 104.7 V / ZNapds1opd Pyazosis RNE 49 46.7 36.5 59.9 ΡΙ (ϋ21) 49 204.2 156.4 266.7 Iiaph RNE 49 46.1 35.3 60.1 ΡΙ (ϋ21) 49 133.7 100.9 177.3 Neither \ L / \ L / RNE fifty 48.6 36,4 64.7 ΡΙ (ϋ21) 49 128.2 101.7 161.6

E1iA303: B1i-vaccine (VEi58A16) in a mixture with adjuvant A803 (E621024A8).

E1iag1x: B1i-vaccine (18854B9).

NNHUUU: E1i ^ UU-vaccine (VESH59A2).

PBE = before vaccination, 14 (1) 21) = 21st day after vaccination.

95% O, PP and BB = 95% confidence interval, lower and upper limits.

Table 12

Antibody Group N Responders 95% CI and υί η % Α / Νβνν Pyazosis 48 25 52.1 37,2 66.7 Sa1ebop1a Iiaph 49 24 49.0 34,4 63.7 Πυνννν 49 eighteen 36.7 23,4 51.7 A / Rapata Pyazosis 48 27 56.3 41.2 70.5 Iiaph 49 23 46.9 32,5 61.7 Pi \ L ^ 49 21 42.9 28.8 57.8 B / bapds! Pyazosis 48 26 54,2 39.2 68.6 Iiaph 49 23 46.9 32,5 61.7 NiU \ LL / 49 sixteen 32,7 19.9 47.5

E1iA803: B1i-vaccine (VBi58A16) in a mixture with adjuvant A803 (B621024A8).

E1iag1x: B1i-vaccine (18854B9).

['1n \ UUU: E1i ^ UU-vaccine (VBSh59A2).

Ν = number of subjects with available results both before and after vaccination. n = number of responders.

% = share of responders (n / Νχ 100).

95% CI = strictly 95% confidence interval; PP = lower limit, Bb = upper limit.

Key Results

Higher CMT and seroconversion levels were observed for hemagglutinin compared with neuraminidase.

CMT antibodies before vaccination for all vaccine strains were in the same range in three groups. Following vaccination, antineuraminidase antibody levels increased significantly. As regards the titers of S-antibodies, after vaccination there was a tendency to higher (values) CMT S-antibodies for all three vaccine strains in the B1iA803 and B1iapkh groups, although there was some overlap of 95% CI between the B1iapkh groups and the Pi ^ UY groups.

- 31 011419

As for the levels of seroconversion, the levels of overall response for the three strains were essentially equal in three groups and for three strains.

The results of the inventors show that healthy elderly people vaccinated in this study against influenza developed good antibody responses to the neuraminidase antigens of any influenza vaccine. However, the response to the neuraminidase antigen is lower than the response to the hemagglutinin antigen.

ΙΙΙ.5.2. Cellular immune response.

Antigen-specific C1-4 and C1-1-8 peripheral blood T cells can be re-stimulated with ΐη νΐΐΐΌ to produce 1b-2, CB40b, аль-alpha and ΙΡΝγ when incubated with the corresponding antigen. As a result, the number of antigen-specific C1-4 and C1-8 T cells can be counted by flow cytometry using traditional immunofluorescence labeling of the cell phenotype, as well as intracellular cytokine production. In the present study, the influenza vaccine virus antigen and peptides of a specific influenza virus protein were used to re-stimulate influenza-specific T cells. The results for C1) 4 and C1) 8 T cell response are presented in table. 13-18.

Table 13

Antigen-specific C1) 4 T-cell responses expressed in cells producing at least two different cytokines: descriptive statistics for time points before (ΡΚΕ) and after vaccination (ΡΟ8Τ) for ΓΡ40Γ / 1Γ2 / ΤΝΡ-α / 1ΡΝ-Secretion γ (whole cohort

СО401_ / 11_2 / ΙΡΝγ / ΤΝΡα in СО4

Antigen Gr. Time point N Average 8ϋ Μΐη Peptides one Day 0 44 33,50 139,026 1.00 the virus one 21st day 45 58.40 132,664 1.00 flu 2 Day 0 42 92.10 368,790 1.00 2 21st day 44 88.36 272,528 1.00 3 Day 0 45 80.13 284,316 1.00 3 21st day 47 91.40 382,967 1.00 Splitting one Day 0 47 1901.66 1596,203 102.00 flaxen one 21st day 48 6163.75 4265,900 773.00 virus 2 Day 0 45 2151.04 2622,594 265.00 flu 2 21st day 49 4150.73 3,712,469 328.00 3 Day 0 48 1678.44 916,329 142.00 3 21st day fifty 3374.60 1920,194 449.00 Whole one Day 0 48 3134.33 2568,369 507.00 virus one 21st day 47 9332.04 6875,403 1482.00 flu 2 Day 0 47 3050.85 2654,936 486.00 2 21st day 49 6760.31 6788,258 1852.00 3 Day 0 48 2955.33 2019,233 473.00 3 21st day fifty 5661.40 4530,321 635.00

Secretion Antigen Gr Time point 01 Median Oz Mach Significant. R* Peptides one Day 0 1.00 1.00 4.00 915.00 the virus one 21st day 1.00 1.00 56.00 733.00 flu 2 Day 0 1.00 1.00 54.00 2393.00 0.7631 2 21st day 1.00 1.00 69.50 1740.00 3 Day 0 1.00 1.00 65.00 1908.00 3 21st day 1.00 1.00 63.00 2615,00 SE40 Splitting one Day 0 957.00 1560.00 2,408.00 9514,00 /! B2 flaxen one 21st day 3468.00 4908.00 7624.00 21324.00 / ΙΡΝγ virus 2 Day 0 930.00 1381.00 2274.00 16289.00 0,0002 / ΤΝΡα flu 2 21st day 2247,00 3036.00 4744,00 21,924.00 in CO2 3 Day 0 1086.00 1502.00 2189,00 3899.00 3 21st day 2312.00 3040.00 4437,00 10431,00 Whole one Day 0 1730.00 2298.50 3876,00 15066,00 virus one 21st day 4091.00 6523,00 14045,00 29251.00 flu 2 Day 0 1190.00 2031.00 4,161.00 11,994.00 0.0040 2 21st day 3573,00 4621,00 7234.00 40173,00 3 Day 0 1421.50 2668.50 3411,50 10,578.00 3 21st day 2459,00 4,315.00 7303.00 22053.00

Group 1: P1iA803: P1i-vaccine Niap ™ mixed with A803 adjuvant.

Group 2: Niap: P1i vaccine Niap ™.

Group 3: Р1и№ УУ: Р1и ννν-vaccine.

8Ό = standard deviation.

Μΐη, Max = minimum, maximum.

C) 1 = first quartile; 03 = third quartile.

Ν = number of subjects with available results.

* P-value: Kruskal-Wallis test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between the three groups on day 21.

- 32 011419

Table 14 Antigen-specific CO2 T cell responses expressed in cells producing at least two different cytokines: descriptive statistics of the difference between time points before (PKE) and after vaccination (PO8T) (entire cohort of vaccinees)

Secretion Antigen Group N Average 8ϋ Μϊη Peptides one 44 9.57 159,363 -860.00 flu virus 2 42 -40.98 386,998 -2392,00 3 45 -50.73 256,596 -1664.00 Οϋ40υΐΡΝ ^ / Split one 47 4307.02 4468,828 -8161.00 ΤΝΡ-α flu virus 2 45 1982.93 3802,332 -14318.00 at Οϋ4 3 48 1555.90 1596,216 -526.00 Whole virus one 47 6197.98 7220,765 -11763.00 flu 2 47 3,791.34 5820,894 -2128.00 3 48 2535.98 3966,345 -4766,00 Peptides one 42 -15.95 215,710 -451.00 flu virus 2 41 50.83 264,370 -614.00 3 44 -52.11 243,811 -684.00 ΟΟ40υΐΡΝ ^ / Split one 42 134.71 426,699 -603.00 ΤΝΡ-α flu virus 2 44 -65.05 822,036 -4938,00 Βθϋ8 3 45 2.49 330,700 -1094,00 Whole virus one 39 189.38 1394,153 -2641.00 flu 2 44 -479.75 1790,094 -9,455.00 3 44 -243.73 719,269 -1892,00

Secretion Antigen Gr. 01 Median Oz Mach Banner R Peptides one 0.00 0.00 37.50 430.00 0,0765 the virus 2 -15.00 0.00 26.00 514.00 flu 3 -37.00 0.00 0.00 212.00 SO40Y Split one 1888,00 3396,00 6634,00 19555.00 <0.0001 ΡΝ · ^ / ny virus 2 699.00 1490.00 2573,00 15169.00 ΤΝΡ-α flu 3 466.00 1183.50 2186.50 7851.00 in CO2 Whole one 2170.00 4009.00 11,681.00 25570.00 0,0003 flu virus 2 1246.00 2382,00 3992.00 33801.00 3 503.00 1382.50 3300.50 19,337.00 Peptides one -106.00 0.00 81.00 655.00 0.0932 the virus 2 -58.00 13.00 202.00 703.00 flu 3 -160.50 0.00 53.00 567.00 SO40Y Split one -122.00 35.50 221.00 1387.00 0.2121 ΡΝ-γ / ny virus 2 -64.50 0.00 160.50 1252.00 ΤΝΡ-α flu 3 -99.00 0.00 76.00 1060.00 in СО8 Whole one -420.00 49.00 591.00 5045,00 0,0851 flu virus 2 -1016.00 -263.50 180.00 3,743.00 3 -651.00 -86.50 180.00 1011.00

Table 15 Antigen-specific CO4 T cell responses expressed in cells producing at least CO401 and another cytokine: descriptive statistics of the difference between time points before (PKE) and after vaccination (PO8T) (entire cohort of vaccinated)

Secretion Antigen Group N Average 8ϋ Μίη Peptides one 44 10.09 153,007 -815.00 flu virus 2 42 -29.40 316,983 -1921.00 3 45 -43.73 251,146 -1629,00 СО401. Split one 46 4266,20 4470,807 -8093,00 in CO2 flu virus 2 45 2026.42 3511,508 -11482.0 3 47 1512.34 1576,133 -494.00 Whole virus one 47 6071.96 7118,132 -11691.0 flu 2 47 3764.64 5740,762 -2114.00 3 48 2544.27 3959,879 -4390.00 Peptides one 44 -19.41 81,675 -370.00 flu virus 2 41 -3.98 100,998 -399.00 3 45 -5.56 64,666 -181.00 СО401- Split one 43 39.53 190,122 -438.00 in СО8 flu virus 2 44 27.61 91,173 -155.00 3 45 30.18 191,326 -291.00 Whole virus one 41 -91.24 617,077 -1779.00 flu 2 44 -115.91 588,424 -2583.00 3 45 -150.89 367,300 -1239,00

- 33 011419

Secretion Antigen Group 01 Median Oz Mach Banner R Peptides one 0.00 0.00 36.50 428.00 0,1233 the virus 2 -8.00 0.00 27.00 494.00 flu 3 -35.00 0.00 3.00 230.00 C0401. Split one 1799.00 3156.50 6647.00 19,480.00 <0.0001 in CO2 ny virus 2 783.00 1485,00 2546.00 15021,00 flu 3 469.00 1107.00 2035.00 7,687.00 Whole one 2109.00 4048.00 11472,00 25,448.00 0,0004 flu virus 2 1212.00 2,509.00 3957,00 33,428.00 3 523.00 1392.00 3261.50 19478,00 Peptides one -2.00 0.00 0.50 100.00 0.9721 the virus 2 -28.00 0.00 24.00 231.00 flu 3 -13.00 0.00 3.00 176.00 СО40Ь Split one -35.00 0.00 140.00 608.00 0.6175 in СО8 ny virus 2 -18.50 0.00 77.50 326.00 flu 3 -9.00 0.00 28.00 1,188.00 Whole one -142.00 -8.00 175.00 2087,00 0.3178 flu virus 2 -195.50 -34.50 150.00 1258.00 3 -270.00 -103.00 88.00 588.00

Table 16 Antigen-specific CT) 4 T-cell responses expressed in cells producing at least ΙΡΝγ and another cytokine: descriptive statistics of the difference between the time points before (PKE) and after vaccination (PO8T) (entire cohort of vaccinated)

Secretion Antigen Group N N no Average 5ϋ Μΐη Peptides one 44 5 7.50 64,539 -171.00 the virus 2 42 7 -30.67 277,984 -1766.00 flu 3 45 5 -27.91 103,403 -639.00 ΙΡΝγ Split one 46 3 2712.87 2905,629 -4394,00 at Οϋ4 ny virus 2 45 4 1148.56 2526,536 -10568.0 flu 3 47 3 871.00 1016,251 -764.00 Whole one 47 2 4240.09 4811,891 -8,272.00 flu virus 2 3 47 48 2 2 2445.38 1535.48 4030,694 2456,915 -3018.00 -3670.00 Peptides one 44 5 7.75 146,412 -226.00 the virus 2 41 8 10.68 176,026 -420.00 flu 3 44 6 -49.80 217,214 -699.00 ΙΡΝγ Split one 43 6 138.58 365,565 -470.00 in СО8 ny virus 2 44 5 -112.82 793,746 -4919,00 flu 3 44 6 29.91 238,157 -708.00 Whole one 41 8 6.66 1642,577 -5610.00 flu virus 2 3 44 44 5 6 -471.55 -189.05 1792,348 685,291 -9586.00 -1879,00

Secretion Antigen Group 01 Median 03 Mach Banner R Peptides one -9.50 0.00 7.50 265.00 0.1541 the virus 2 -5.00 0.00 24.00 222.00 flu 3 -20.00 0.00 0.00 51.00 ΙΡΝγ Split one 1273,00 1644,00 4057.00 13,296.00 <0.0001 at Οϋ4 ny virus 2 405.00 931.00 1757.00 9426.00 flu 3 283.00 624.00 1114.50 5031,00 Whole one 1610.00 2693,00 7,437.00 17489.00 <0.0001 flu virus 2 723.00 1487.00 2983.00 21,594.00 3 232.50 810.00 2218.50 11319,00 Peptides one -52.50 0.00 40.00 615.00 0.3322 the virus 2 -1.00 0.00 72.00 610.00 flu 3 -172.00 0.00 90.50 424.00 ΙΡΝγ Split one -46.00 42.00 294.00 1549.00 0.1257 in οϋδ ny virus 2 -62.00 0.00 74.00 1028.00 flu 3 -59.50 26,50 123.00 643.00 Whole one -385.00 131.00 450.00 5068,00 0.1179 flu virus 2 -955.50 -221.00 177.00 3492,00 3 -476.50 -36.50 198.00 1299.00

- 34 011419

Table 17 Antigen-specific CO2 T cell responses expressed in cells producing at least II .2 and another cytokine: descriptive statistics of the difference between time points before (PKE) and after vaccination (PO8T) (entire cohort of vaccinated)

Secretion Antigen Group N Average 5ϋ Μϊη Peptides one 44 2.82 118,164 -595.00 the virus 2 42 0.90 84,255 -167.00 flu 3 45 -28.62 191,709 -1222,00 11_2 Split one 46 3456.15 3853,960 -7009.00 in CO2 ny virus 2 45 1738.29 2406,045 -451.00 flu 3 47 1210.02 1361,705 -634.00 Whole one 47 4839,02 5978,277 -9178.00 flu virus 2 47 2891.00 4,493,387 -1370.00 3 48 2042,50 3123,912 -3179.00 Peptides one 42 -30.60 219,777 -630.00 the virus 2 41 38.85 210,715 -674.00 flu 3 45 -44.80 197,026 -526.00 11_2 Split one 41 54.85 250,817 -336.00 in СО8 ny virus 2 44 -2.36 423,957 -2272,00 flu 3 45 -26.07 244,870 -1004.00 Whole one 39 56.21 406,262 -704.00 flu virus 2 44 -151.02 822,384 -4,304.00 3 45 -63.56 359,699 -1036.00

Secretion Antigen Group 01 Median Oz Mach Significant. R Peptides one -1.50 0.00 31,50 324.00 0,0806 the virus 2 -34.00 0.00 2.00 362.00 flu 3 -19.00 0.00 0.00 253.00 ΙΙ_2 Split one 1309,00 2598.50 5926.00 16988,00 <0.0001 in CO2 ny virus 2 453.00 1113.00 2049.00 12273,00 flu 3 331.00 806.00 1596,00 6474,00 Whole one 1516.00 3341,00 8955.00 21032,00 0,0006 flu virus 2 995.00 1942.00 3007.00 26,358.00 3 371.50 1083,50 2624,50 14057,00 Peptides one -111.00 0.00 103.00 412.00 0.1684 the virus 2 -41.00 0.00 138.00 542.00 flu 3 -150.00 -34.00 71.00 447.00 11_2 Split one -76.00 26.00 133.00 803.00 0.2311 in СО8 ny virus 2 -78.50 0.00 121.50 1064,00 flu 3 -93.00 -1.00 30.00 705.00 Whole one -167.00 63.00 261.00 1302.00 0.4586 flu virus 2 -444.50 -4.00 199.00 1398,00 3 -198.00 9.00 131.00 838.00

Table 18 Antigen-specific CO2 T cell responses expressed in cells producing at least ΨΝΕα and another cytokine: descriptive statistics of the difference between time points before (PKE) and after vaccination (PO8T) (entire cohort of vaccinated data)

Secretion Antigen Group N Average Μίη Peptides one 44 9.48 92,992 -466.00 the virus 2 42 -47.71 367,624 -2333.00 flu 3 45 -37.38 179,147 -1169,00 ΤΝΡ-α Split one 46 2343.11 2596,177 -4450.00 _ L in ny virus η L s * + and (oo, o / ALTP P A A Ο, ΖΗ 1 A a - ι ^ ζου, υ flu 3 47 732.00 740,001 -611.00 Whole one 47 3103.74 4248,997 -5146.00 flu virus 2 47 1658.38 3639,959 -1393.00 3 48 1010.15 1689,394 -1482.00 Peptides one 42 11.71 201,031 -453.00 the virus 2 41 37.46 245,241 -612.00 flu 3 44 -42.95 210,185 -645.00 ΤΝΡ-α Split one 41 138.54 362,601 -329.00 in СО8 ny virus 2 44 -70.27 790,309 -4741,00 flu 3 44 -39.75 348,803 -1044,00 Whole one 39 279.59 1048,352 -1184,00 flu virus 2 44 -280.70 1562,095 -9070.00 3 44 -71.57 492,135 -1574.00

- 35 011419

Secretion Antigen Group 01 Median Oz Mach Significant. R Peptides one -1.50 0.00 39.00 239.00 0.1836 the virus 2 -4.00 0.00 12.00 277.00 flu 3 -26.00 0.00 5.00 53.00 ΤΝΕ-α Split one 862.00 1466.50 3931.00 9267.00 <0.0001 in CO2 ny virus 2 251.00 698.00 1229,00 12,275.00 flu 3 191.00 540.00 1010.00 3288,00 Whole one 868.00 1607.00 5266.00 17199.00 0,0008 flu virus 2 367.00 871.00 1584,00 23540.00 3 175.00 592.00 1385.50 8760.00 Peptides one -80.00 0.50 70.00 772.00 0.2759 the virus 2 -81.00 0.00 155.00 791.00 flu 3 -179.00 0.00 39.50 566.00 ΤΝΕ-α Split one -23.00 60.00 178.00 1468.00 0,0790 in СО8 ny virus 2 -107.00 0.00 158.00 1286.00 flu 3 -185.00 0.00 78.50 1021,00 Whole one -250.00 108.00 399.00 4,601.00 0.1482 flu virus 2 -392.00 -56.50 205.00 3258,00 3 -233.50 -54.00 160.00 1543,00

The results were also expressed as the frequency of occurrence of cytokine (s) -positive CO4 or CO8 T cells among C1) 4 or CO8 T cell subpopulations, and are shown in FIG. 4 and 5.

In a similar analysis, a cross-reactive CO4 T cell response was evaluated using an influenza virus antigen from drifting strains (Α / Η1Ν1 / Бе1†1пд/262./95 (Η1Ν1ά), ΑΗ3Ν2 Tuyneu / 5, / 97 (Η3Ν2ά), Β / Υатаηа / 166/98 (Βά)) or shift strains (A / 8taroge / 1/57 (Η2Ν2), A / Lopdkopd / 1073/99 (Η9Ν2)). The results, expressed as the frequency of occurrence of cytokine (s) -positive CO4 cells, are presented in FIG. 6.

Key Results

Vaccination with E1iapch or whole virus slightly activates the CO4 T-cell response. Vaccination with E1nL8 () 3 induces a strong CO4 T cell response (Fig. 4), and this result is statistically significant. The same conclusion is made after stimulation with ΐη νίΐΐΌ by a split antigen or whole virus, and this is observed for all the cytokines studied (III-2, ΙΕΝγ, ΤΝΕα and CE40b).

Most individuals have a CE8 T-cell response to whole Mon, however, vaccination does not have a measurable effect on the CE8 T-cell response (i.e., IgeOee!) For any large studied (Fig. 5).

Vaccination only IIAPh induces low levels of cross-reactive CE4 T-cell response (Fig. 6). Vaccination n1L803 induces a strong CE4 T cell response to drifting strains of the influenza virus, and this result is statistically significant (Fig. 6). Against shift strains, the response was negligible.

ΙΙΙ.5.3. EShroEanalysis of B-cells of memory.

ΙΙΙ.5.3.1. Task.

In order to better characterize the CΜI response induced by the A803 adjuvanted influenza vaccine, the ESOE analysis evaluated the B-cell secondary immune response induced to differentiate into ΐη νϊΐΐΌ plasma cells using anti-influenza vaccine strains or an anti-human immunoglobulin cell antibody for secreting antibodies against influenza virus or ΙΑ.ι. The results are described in table. 19 and 20 and in FIG. 7.

A subgroup consisting of the first 22 subjects who received 1 dose of the E1nL803 vaccine and of the first 21 subjects who received 1 dose of the E1iap vaccine were selected to assess the effect of vaccination on influenza virus-specific memory B cells using ESroE technology for evaluating Β-cell memory. The following endpoints were determined.

On days 0 and 21: influenza virus-specific memory B cells were measured using an E-ChrEanalysis of B cells in all subjects. The results were expressed as the frequency of occurrence of cells forming influenza virus-specific antibodies per million (10 ⁶ ) antibody-forming cells.

The difference between the conditions after (21st day) and before (0th day) vaccination was also expressed as the frequency of occurrence of cells that form antibodies specific for the influenza virus per million (10 ⁶ ) antibody-forming cells.

ΙΙΙ.5.3.2. Statistical methods.

Descriptive statistics for each vaccinated group on days 0 and 21, expressed as the frequency of occurrence of cells that form antibodies specific for the influenza virus, per million (10 ⁶ ) antibody-forming cells. Descriptive statistics of the individual difference between the 21st and 0th days (yoMeite ') in the form of the frequency of occurrence of cells that form antibodies specific for the influenza virus per million (10 ⁶ ) antibody-forming cells.

To compare the differences found between the two groups, the Wilcoxon test was used and the statistical value p was calculated for each of the three strains (L. 'et al Sáebosha, A / Gapata and й Tianuyopu).

ΙΙΙ.5.3.3. Results.

There is a trend in favor of the influenza A803 adjuvant vaccine compared to the E1iag1x group. For strain Α / Νβ ^ Ca1ebosh there is a statistically significant difference (p = 0.021) in

- 36 011419 favor of P1iA803 in comparison with P1iapkh. As for strains A / Rapata and B / 8Pap§bop§, no statistical difference between the two groups was observed.

Table 19

B-cell memory: descriptive statistics for time points before (day 0) and after (day 21) vaccination and output statistics for the frequency of occurrence after vaccination (day 21) of antigen-specific plasma cells among 10 ⁶ Ι ^ Θ-producing plasma cells (subgroup of subjects)

STRAIN Group Time point N Average 5ϋ Μϊη Α / ΝΕνν SAJEOOMA one Day 0 22 9751.58 6630,335 0.00 one 21st day 22 22001.65 11308,261 3981.90 2 Day 0 21 9193.61 4339,421 1300.81 2 21st day 21 12263.08 7,285,698 789.47 .Α / ΡΑΝΑΜΑ one Day 0 22 4329.17 2923,497 0.00 one 21st day 22 18066.69 14,604,842 714.29 2 Day 0 21 4860.41 3392,373 0.00 2 21st day 21 13872.95 12052,163 0.00 Β / 8ΗΑΝΟΟΟΝΟ one Day 0 22 3722.80 2347,315 0.00 one 21st day 22 15,949.60 12385,965 0.00 2 Day 0 21 3030.39 2206,589 640.57 2 21st day 21 9714.03 5656,805 0.00

STRAIN Gr Time point 01 Median Oz Mach Significant. R* Α / ΝΕνν ΟΑΙ, ΕΟΟΝΙ A one Day 0 4117.65 9606.46 13430.66 25570.78 0.0056 one 21st day 11052.63 20,450.55 30,234.74 40526.32 2 Day 0 6363.64 9686.41 11698.11 19164.84 2 21st day 7741.05 9545.45 17069.60 32,000.00 Α / ΡΑΝΑΜΑ one Day 0 2275.45 4003.02 5764.55 10,842.49 0.1814 one 21st day 9347.37 13176.41 21,471.39 54789.92 2 Day 0 2222.22 4545.45 7495.74 11698.11 2 21st day 6,231.88 10147.06 20540.54 52,188.84 Β / 8ΗΑΝΟϋΟΝΟ one Day 0 2058.82 2956.78 5972.22 7832.17 0.1483 one 21st day 6860.47 12796.90 22947.37 48,947.37 2 Day 0 1290.32 2113.82 4770,02 7783.25 2 21st day 6590.91 9009,01 12774.87 21201.72

Group 1: P1i-vaccine P1iaph ™ + A803 adjuvant in the form of an oil-in-water emulsion.

Group 2: P1i-vaccine P1iaph ™.

8Ό = standard deviation.

Mt, Mach = minimum, maximum.

C) 1 = first quartile.

C) 3 = third quartile.

N = number of subjects with available results.

P * value: Wilcoxon test (nonparametric technique) for testing the detected difference (Wilcoxon rank sum test) between the two groups on day 21.

Table 20

B-cell memory: descriptive and statistics of the conclusion for the difference between time points after (21st day) and before (0th day) vaccination according to the frequency of occurrence of antigen-specific plasma cells among 10 ⁶ 1c6-producing plasma cells (subgroup of subjects)

STRAIN Group Ν Average δϋ Μίη Α / ΝΕνν ΟΑΙ ΕΟΟΝΙΑ one 22 12,250.07 12875,7555 -4365.08 2 21 3069,46 7309,731 -10043,4 Α / ΡΑΝΑΜΑ one 22 13737.52 13677,942 -188.29 2 21 9012.54 11489,012 -1551.05 Β / δΗΑΝΘϋΟΝΟ one 22 12226.81 12,243,895 -2222.22 2 21 6683.64 6,240.312 -2113.82

STRAIN Γρ. οι Median 03 Mach Significant. ρ (Wilcoxon test) .Α / ΝΕνν ΟΑΙ ΕϋΟΝΙΑ one 2418.07 6776.65 26036,01 35059.98 0.0210 2 -1762.54 1694.51 6850.19 18,579.97 / Α / ΡΑΝΑΜΑ one 4551.30 11039.04 16614.85 49881.94 0.1449 2 1522.85 6480.96 9214.67 47812.47 Β / 8ΗΑΝΘϋΟΝΘ one 1788.75 9322.70 18907.05 42,134.18 0.1895 2 2117.44 5384.41 9897.27 19801.28

Group 1: P1i-vaccine P1iaph ™ + A803 adjuvant in the form of an oil-in-water emulsion.

Group 2: P1i-vaccine P1iaph ™.

- 37 011419

8Ό = standard deviation.

M1i, Max = minimum, maximum.

C) 1 = first quartile. 03 = third quartile.

N = number of subjects with available results.

P-value: Wilcoxon test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between the two groups on day 21.

S.6. General conclusions.

S.6.1. Reactogenicity and safety results.

Despite the fact that immunization with the influenza virus significantly reduces the risk of pneumonia and its associated deaths, vaccination of older people provides only 23-72% protection against influenza. Vaccine antigen preparation technology with effective adjuvants is an attractive approach to enhancing immune responses to subunit antigens. This study was designed to evaluate: (1) the safety and reactogenicity of an influenza vaccine with adjuvant in the form of an oil-in-water emulsion, i.e. A803, in healthy older adults, (2) antibody and cell-mediated immune responses. Reactogenicity data indicate that the influenza vaccine with A803 adjuvant induced more local and general symptoms than the other two vaccines. However, with regard to unforeseen adverse events, no difference between the three vaccines was observed. From these results, it can be concluded that the reactogenicity and safety profile of candidate vaccines is satisfactory and clinically acceptable.

S.6.2. Immunogenicity results.

As regards the immune response, the three vaccines exceeded the requirements of the European authorities for the annual registration of split virion influenza vaccines ) The three influenza vaccines tested in this study were immunogenic for healthy elderly people who developed a good antibody response to influenza virus hemagglutinin and neutralizing antigens (Table 21).

Table 21

Variable EU standard for antibody response results Conversion factor > 2.0 > 6.1 Seroconversion rate > 30% > 50% Protection level > 60% > 88%

As for the cell-mediated immune (SMH) response, the influenza vaccine with A803 adjuvant induced a significantly stronger CE4 response (including drifting strains) than the other two vaccines (P1 and Apo and whole-virus influenza vaccine). However, vaccination did not have a measurable effect on the CE8 response.

As for the B-cell secondary immune response, there is a tendency in favor of an influenza adjuvant vaccine compared to a non-adjuvant vaccine.

Example IV A clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus and A803 adjuvant in an elderly population over the age of 65 is Exp1o-P1i-002.

Phase VII of an open controlled study was carried out to assess the reactogenicity and immunogenicity of the influenza vaccine candidate candidate for O1axo8tDGK1te Vu1od1ca1z containing adjuvant A803, in an elderly population over 65 years old, previously vaccinated in 2003. candidate vaccine in a clinical trial Exp1o-P1i-001. To evaluate immunogenicity and safety, the Niapx ™ vaccine was used for comparison (known in Belgium as a-K1x ™).

ΣV.1. Task.

The humoral immune response (i.e., anti-hemagglutinin antibody titers), and the cell-mediated immune response (CT) 4 and / or CT) 8 T-cell responses) and the B-cell secondary immune response were measured 21 days after intramuscular administration 1 doses of A803 adjuvant vaccine. Niap ™ was used for comparison.

The tasks were set:

1) determine whether the fact that A803-adjuvant P1i (40 subjects) in comparison with Niapch (18 subjects) has the highest immunostimulatory activity against CT) 4- and / or CE8-mediated immunity in individuals vaccinated with influenza antigens is confirmed ;

2) to study, using long-term analysis, the effect of the addition of A803 adjuvant on the immune response before vaccination in 2004. (i.e. response 1 year after the first vaccination in 2003).

! U.2. Study design, vaccine composition, and endpoints.

Subjects over the age of 65 who have previously received 1 dose of A803 adjuvant

- 38 011419 flu vaccines during a clinical trial of Exp1o-E1i-001 in 2003. (Η1γιΑ§03).

One control group of approximately 20 subjects over the age of 65 who previously received 1 dose of Niapx ™ during a 2003 clinical trial of Exp1-E1i-001. (Niaph).

Ιν.2.1. Vaccine composition.

The vaccine composition is similar to that used for the study of Exp1-E1i-001, with the exception of the influenza virus strains included in the vaccine (2004 vaccine). The following strains are used:

Α / No. \ ν Ca1ebosha / 20/99 (ΙνΚ-116) (Η1Ν1) - a strain similar to Α / No. \ ν Ca1ebosha (ΗΙΝΙ); АЖуоттд / 3/2003 (Х-147) (Η3Ν2) - a strain similar to A / Ecstsap (Η3Ν2);

B / Lap§8c / 10/2003 is a strain similar to B / 8yapdya1.

Ιν.2.2. Endpoints for immunogenicity (ΗΙ).

SMΤ (taking the antilogarithm of the mean for the logarithmic transformations of the titles).

Conversion factors (a multiple increase in serum stMH ΗΙ on the 21st day compared to the 0th day). Seroconversion level (percentage of vaccinees having at least four-fold increase in увеличения-titers on the 21st day compared to 0 days for each vaccine strain).

Level of protection (percentage of vaccinees with serum тит-titer greater than or equal to 1:40 on the 21st day).

Ιν.2.3. Endpoints for CME

Observed variable

On day 0 and day 21: the frequency of occurrence of cytokine-positive CO4 / SE8 cells by 10 ⁶ for four different cytokines. In each test, the response of ί.Ό4 / ί.Ό8 T cells to the pool of the following three antigens was quantified;

antigen No. \ ν Ca1ebosha;

HUuotnsch antigen;

Lapdka antigen.

Secondary variables

Antigen-specific CE4 and SE8 T-cell response, expressed in five different tests (cytokines):

1) cells producing at least two different cytokines (CE40b, ΙΕ-2, ΙΡΝγ, ΤΝΡα);

2) cells producing at least CE40b and another cytokine (ΙΕ-2, ΤΝΡα, ΙΡΝγ);

3) cells producing at least ΙΕ-2 and another cytokine (CE40b, ΤΝΡα, ΙΡΝγ);

4) cells producing at least ΙΡΝγ and another cytokine (ΙΕ-2, ΤΝΡα, CE40b);

5) cells producing at least ΤΝΡα and another cytokine (ΙΕ-2, CE40b, ΙΡΝγ).

Ιν.2.4. CME analysis

The first SM analysis was performed for the entire cohort of vaccinees (Ν corresponds to 40 subjects for the Ρ1υΑ803 group, and Ν corresponds to 18 subjects for the Ρ1ia ^^ x group).

A long-term analysis was carried out for a dynamic cohort in the studies of Exp1o-E1i-001 (digested protein) and Exp1o-E1i-002 (Di-antigen pool):

Rge: Ν corresponds to 36 subjects for the уп1υΑ803 fuppa, and Ν corresponds to 15 subjects for the Ρ1ia ^^ x fuppa; Po51-Rge: Ν corresponds to 34 subjects for the group Ρ1ηΑ803. and Ν corresponds to 15 subjects for the group Ρ1ia ^^ x.

(a) The frequency of occurrence of CO4 / CO8 β-lymphocytes secreting in response was summarized using descriptive statistics for each antigen, for each cytokine, for each vaccine group, and for each time point (before and after vaccination).

(b) Data from descriptive statistics of the individual differences between responses to time points (Po & Pr) were presented in tabular form for each antigen, for each cytokine and for each vaccine group.

(c) For the time points corresponding to after (ro51) and after-before (ro51-rge) vaccinations, the non-parametric Wilcoxon test was used to compare the differences found between the two vaccine groups and to calculate the statistical value of p related to four different cytokines,

ί.Ό4 T-cell response to No. \ ν Ca1ebosha, HUuopnpd. Lapdki and pool of three strains;

ί.Ό8 T-cell response to No. \ ν Ca1ebosha, HUuopnpd. Lapdki and pool of three strains.

(d) A nonparametric test (Wilcoxon test) was also used to study the kinetics of the immune response for the (time point) Pg (day 0) in relation to the frequency of occurrence of specific CE4 (cells) between Exp1o-E1i-001 and Exp1o-E1i -002 in each vaccine group;

to study the kinetics of the immune response for the (time point) PGE (day 0) in relation to the frequency of occurrence of specific CE4 (cells) between two vaccine groups in each study Exp1o-E1ts-001 and Exp1o-E1ts-002;

to study the kinetics of the immune response in relation to the differences (PoCh-PTE) in the frequency of occurrence of specific CE4 (cells) between Exp1o-E1i-001 and Exp1o-E1i-002 in each vaccine group;

to study the kinetics of the immune response in relation to the differences (Po51-Prz) in the frequency of occurrence of specific CE4 (cells) between two vaccine groups in each study Exp1o-E1i-001 and Exp1o-Ρ1i-002.

All criteria of significance were bilateral. P values of less than or equal to 0.05 were considered statistically significant.

- 39 011419

GU. 3. Results.

The results were expressed as the frequency of occurrence of cytokine (s) -positive CO4 or CO8 T cells among C1) 4 or CO8 T cell subpopulations.

UAZD. Antigen-specific CO2 T cells.

The frequency of occurrence of antigen-specific C1) 4 T-lymphocytes secreting in response was summarized using descriptive statistics for each antigen, for each cytokine, for each vaccine group, and for each time point (before and after vaccination).

Descriptive statistics of the individual differences between time points (RosERge) in the CO2 responses of T lymphocytes for each antigen for each five different cytokines and for each vaccine group are shown in Table. 22.

Table 22

Descriptive statistics of the difference between the time points after vaccination (on the 21st day) and before vaccination (on the 0th day) for the responses of antigen-specific CO4 T lymphocytes (the entire cohort of vaccinated)

Antigen Cytokine Vaccine group N Avg 813 M1p 01 Median az Mach Pool pi All two P1iag1kh NI AZOZ 16 3 £ 1268.67 1781.31 1051,744 1484,860 197.00 2379.00 724.00 929.50 863.00 1664.50 1561,00 2821,00 4676,00 4669,00 С040Ь Р1иаг1х Р1и АЗОЗ 16 3 £ 1260.11 1711.56 1054,487 1433,113 243.00 -2359.00 721.00 838.00 849.00 1576.00 1602.00 2759.50 4743,00 4576.00 ΙΡΝγ R1iapkh NI AZOZ 1E 762.94 1179.92 813,884 881,255 12.00 -617.00 294.00 692.50 496.00 1180.50 1061,00 1865.50 3564,00 2831.00 11.2 P1iag1kh NI AZOZ 18 s 1019.06 1423.33 917,905 1359,471 -258.00 2702.00 544.00 651.00 702.00 1260.00 1174.00 2200.50 3850.00 4,342.00 ΤΝΡα P1iag1kh NI AZOZ eighteen 36 803.39 1078.28 915,838 1029,122 32.00 -1816.00 231.00 446.00 533.00 983.00 936.00 1836.00 3892,00 3310,00 Α / Νβνν Ca1e0op1'a All two Niaph Р1и АЗОЗ eighteen 36 481.44 812.78 381,534 749,192 -241.00 -828.00 282.00 215.50 448.50 911.50 598.00 1274.50 1412.00 3206.00 СО401. P1iag1kh NI AZOZ eighteen 36 450.78 783.75 360,378 711,608 239.00 -760.00 291.00 242.00 447.00 808.00 580.00 1161.00 1248.00 3,050.00 ΙΡΝγ P1iag1kh NI AZOZ eighteen 36 316.28 438.22 279,662 420,770 -165.00 -685.00 175.00 125.00 259.00 393.00 387.00 733.50 1111.00 1557,00 11.2 Р1иаг1х Р1и АЗОЗ eighteen 36 326.06 634.72 290,792 616,478 294.00 -557.00 193.00 179.50 330.00 678.50 488.00 952.00 834.00 2,602.00 ΤΝΡα R1iapkh NI AZOZ eighteen 36 316.44 449.17 372,492 591,736 140.00 -916.00 50.00 100.50 278.00 343.50 542.00 848.00 1449.00 2452.00 AM / wat1pd All two P1iag1kh None Azozoz eighteen 36 609.56 766.61 559,396 579,191 -176.00 -568.00 257.00 316.00 510.50 864.50 957.00 1221.00 1998,00 1662.00 СО401. R1iaph R1i AZOZ eighteen 36 616.33 728.61 550,853 570,316 -176.00 -670.00 274.00 260.00 488.00 789.50 939.00 1216.00 2017.00 1675.00 ΙΡΝγ ΡΙιιβΓΪχ P1 and AZOZ eighteen 36 407.06 526.72 424,758 443,938 -311.00 770.00 129.00 219.00 370.50 556.50 723.00 776.00 1372.00 1342.00 11.2 P1iag1kh NI AZOZ eighteen 36 495.83 572.89 503,805 533,728 -187.00 789.00 88.00 220.00 540.50 602.00 801.00 882.50 1841.00 1512.00 ΤΝΡα R1iapkh NI AZOZ eighteen 36 424.56 550.58 485,591 538,461 -260.00 -765.00 110.00 269.50 359.50 543.50 461.00 905.50 1718.00 1678,00 VLNalji All two P1iaph Neither AZOZ eighteen 36 698.44 861.42 793,119 688,852 -306.00 -223.00 233.00 339.00 433.00 745.00 961.00 1325.50 2822.00 2284.00 3040 Niap Ni Ni Azoz eighteen 36 678.39 825.89 777,259 674,879 -206.00 -223.00 227.00 305.00 401.50 722, OS 962.00 1282.00 2878,00 2337.00 ΡΝγ Niap Pi Azoz eighteen 36 431.72 615.94 489,912 473,543 -95.00 -286.00 191.00 288.50 272.5C 501.50 382.00 897.50 1712.00 1740.00 B2 P1iag1kh NI AZOZ eighteen 36 552.50 696.19 666,853 622,931 -234.00 -359.00 155.00 207.50 278.50 540.50 833.00 1146.50 2386.00 2182.00 ΓΝΡα Niap Ni Ni Azoz eighteen 36 441.39 500,03 695,792 448,636 -338.00 -166.00 97.00 107.50 269.50 436.00 564.00 745.00 2440.00 1626.00

- 40 011419

8Ό = standard deviation.

Μΐη, Max = minimum, maximum.

C) 1 = first quartile.

C) 3 = third quartile.

N = number of subjects tested with available results.

It has been shown that vaccines C1-4 T cells induced can persist for at least 1 year, since there is a noticeable difference in C1-4 T-cell responses before vaccination between individuals vaccinated with P1iapx compared with those vaccinated with P1iag1x / A803 earlier. The results are also shown in FIG. 8, which shows C1) 4 T-cell response to the cleaved P1i antigen before and after revaccination. Ό0 corresponds to 12 months after vaccination of the first year and thus indicates persistence.

When comparing the differences in the frequency of occurrence of antigen-specific C1-4 T cells after vaccination between the two groups according to the Wilcoxon test, it can be seen that almost all p values were less than 0.05 and were considered statistically significant (see Table 23) in favor of the group P1iA803.

Table 23

Conclusion statistics: p values according to the criterion of the sum of Wilcoxon ranks between two vaccine groups on the 21st day for responses of antigen-specific CP4 T lymphocytes (the entire cohort of vaccinated)

Cytokine P value Pool Νθνν Ca1e6ot‘a Wowatt Chaldeans All two 0.0014 0.0023 0,0286 0.0133 СО401- 0.0016 0.0014 0.0427 0.0155 ΙΝΕγ 0,0006 0,0366 0,0400 0.0041 11 2 0.0037 0.0024 0.0584 0.0162 ΤΝΕα 0.0031 0.0103 0.0918 0.0114

P-value: Wilcoxon test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between the two groups on day 21.

When comparing the difference in individual differences (Ροδΐ-Rhs) in the frequency of response of antigen-specific C1) 4 T lymphocytes between the two groups according to the Wilcoxon test, it is seen that p values less than 0.05 and considered statistically significant were observed for the following antigen-cytokine combinations: pool And and - '' all in two, pool yi-ΙΡΝγ and .Ιίί ·· ιηρ8υ-ΙΡΝγ in favor of the group P1iA803 (Table 24).

Table 24

Conclusion statistics: p values calculated according to the Wilcoxon rank sum criterion between different groups with respect to the difference between the time points after vaccination (on day 21) and before vaccination (on day 0) for antigen-specific responses C1) 4 T lymphocytes (whole cohort of vaccinated)

Cytokine P value Pool Νβνν Ca1ec1ota №wottd Chaldeans All two 0.0435 0.1124 0.2189 0.3085 СО40Ь 0.0638 0,0781 0.2831 0.2872 ΙΝΕγ 0.0290 0.3589 0.2553 0.0435 ΙΙ 2 0.1024 0,0563 0.3986 0.0435 ΤΝΕα 0,0693 0.4090 0,1232 0.3129

P-value: Wilcoxon test (non-parametric method) for testing the detected difference (Wilcoxon rank sum test) between the two groups.

ΐν.3.2. Antigen-specific C1) <8 T-lymphocytes.

The frequency of occurrence of antigen-specific C1) <8 T-lymphocytes secreting in response was summarized using descriptive statistics for each antigen, for each cytokine, for each vaccine group and for each time point (before and after vaccination) by analogy with the methodology for C1 ) 4 T cell responses.

When comparing the differences in the frequency of occurrence of antigen-specific C1) 8 T-lymphocytes after vaccination between the two groups according to the Wilcoxon test, it is seen that all p values exceeded 0.05 and were not considered statistically significant. When comparing the difference in individual differences (Ροδΐ-Rhs) in the frequency of response of antigen-specific C1) <8 T lymphocytes between the two groups according to the Wilcoxon test, it is seen that all p values exceeded 0.05 and were not considered statistically significant.

ΐν.3.3. Kinetic analysis: immune response before vaccination (1 year after the first vaccination in 2003)

Frequency of antigen-specific C1) 4 T-lymphocytes secreting in response

- 41 011419 vaccinations were summarized using descriptive statistics for each cytokine, and for each vaccine group, and for each of the two studies in the table. 25, for each of the two studies and for each vaccine group in the table. 27. The output statistics are presented in table. 26 and 28.

Table 25 Descriptive statistics before vaccination (day 0) for the response of specific CP4 T lymphocytes to vaccination (kinetics)

Cytokine Group Study N Avg $ 0 M ('p Joint venture Copper ana Oz Mach All two None Azozoz EXTR 001 EXTR 002 36 36 2000.86 2028.28 1783,474 1,427,000 102.00 55.00 911.50 1190.50 1461.50 1647.50 2791.00 2575,00 9514,00 7214.00 Yayaph ECRY 001 ECRY 002 fifteen fifteen 2152.87 1587.07 2162,463 2123,841 747.00 192.00 930.00 468.00 1354.00 735.00 2101.00 1578,00 7868,00 8536.00 S0401- None Azozoz ECRY 001 ECRY 002 35 35 1946.66 1992,20 1771.102 1440,721 120.00 77.00 837.00 1125.00 1340.00 1590.00 2819.00 2587,00 9462.00 7286.00 Yayaph EXTR 001 EXTR 002 fifteen fifteen 2094.93 1561.73 2076,632 2097,201 745.00 34.00 902.00 475.00 1340.00 672.00 2077,00 1579,00 7385.00 8428,00 ΙΝΕγ None Azozoz ECRY 001 ECRY 002 35 35 1068.63 1259.23 1030,745 890,590 91.00 312.00 448.00 725.00 790.00 984.00 1503.00 1354.00 5425.00 4146.00 Yayaph EXTR 001 EXTR 002 fifteen fifteen 1248.07 974.80 1452,459 1394,044 320.00 52.00 388.00 252.00 778.00 337.00 1227.00 1057,00 5431.00 5576.00 11-2 None Azozoz ECRY 001 ECRY 002 35 35 1690.20 1883.60 1524,689 1361,337 37.00 14.00 688.00 1068.00 1211.00 1413.00 2416.00 2370.00 8235.00 6891,00 Yayaph EXTR 001 EXTR 002 fifteen fifteen 1888.40 1493.93 2085,857 2037,139 568.00 58.00 715.00 444.00 1136.00 755.00 1770.00 1405.00 7,403.00 8,193.00 ΤΝΕα None Azozoz ECRY 001 ECRY 002 35 35 1174.74 1545.40 1119,633 1159,490 55.00 135.00 466.00 831.00 795.00 1203.00 1720.00 1857.00 5415.00 5,354.00 YaiaNh EXTR 001 EXTR 002 fifteen fifteen 1444.20 1304.73] 1946,211 1759,716 201.00 144.00 520.00 316.00 668.00 824.00 1254.00 1171.00 7213.00 7056.00

ΒΙ) = standard deviation.

M1n, Max = minimum, maximum.

C) 1 = first quartile.

C) 3 = third quartile.

N = number of subjects tested with available results.

When comparing the differences in the frequency of occurrence of antigen-specific C1-4 T cells between two studies according to the Wilcoxon test for each vaccine group, it is seen that p values less than 0.05 and considered statistically significant (in favor of Exp1o-P1i-002) were observed only for the P1iA803 group and with the cytokine ΤΝΕα (see table 26).

Table 26

Conclusion statistics: p values according to the Wilcoxon rank sum criterion between different studies on day 0 for antigen-specific responses C1) 4 T-lymphocytes (kinetics)

Cytokine Group P value ALL FOR TWO Yaiazoz 0,5209 Yayaph 0,0712 СО40Ь Yaiazoz 0.4957 Yayaph 0,0744 ΙΝΕγ Yaiazoz 0,0896 Yayaph 0,1103 ΙΙ_2 Yaiazoz 0.1903 Yayaph 0.1647 ΤΝΕα Yaiazoz 0.0427 Yayaph 0.5476

P-value: Wilcoxon test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between the two groups on day 21.

- 42 011419

Table 27 Descriptive statistics before vaccination (day 0) for the response of specific CP4 T lymphocytes to vaccination (kinetics)

Cytokine Study Group N Avg fifty Μίη 01 Median Oz Mach All two ECRY 001 Р1и АЗОЗ Р! Иag1х 36 fifteen 2000.66 2152.87 1783,474 2162,463 102.00 747.00 911.50 930.00 1461.50 1354.00 2791.00 2101.00 9514,00 7868,00 ECRY 002 Р1и АЗОЗ Р! Иag1х 36 fifteen 2028.28 1587.07 1,427,000 2123,841 55.00 192.00 1190.50 468.00 1647.50 735.00 2575,00 1578,00 7214.00 8536.00 С040Ь ECRY 001 Р1и АЗОЗ Niaph 35 fifteen 1946.66 2094.93 1771.102 2076,632 120.00 745.00 837.00 902.00 1340.00 1340.00 2819.00 2077,00 9462.00 7385.00 ECRY 002 Р1и АЗОЗ Р1иапх 35 fifteen 1992,20 1561.73 1440,721 2097,201 77.00 34.00 1125.00 475.00 1590.00 672.00 2587,00 1579,00 7286.00 8428,00 ΙΝΡγ ECRY 001 ΡΙυ AZOZ R1iag! X 35 fifteen 1068.83 1248.07 1030,745 1452,459 91.00 320.00 448.00 388.00 790.00 778.00 1503.00 1227.00 5425.00 5431.00 ECRY 002 Р1и АЗОЗ Р1иапх 35 fifteen 1259.23 974.80 890,590 1394,044 312.00 52.00 725.00 252.00 984.00 337.00 1354.00 1057,00 4146.00 5576.00 AND 2 ECRY 001 ΡΙυ AZOZ R1iapkh 35 fifteen 1690.20 1888.40 1524,689 2085,857 37.00 568.00 688.00 715.00 1211.00 1136.00 2416.00 1770.00 8235.00 7,403.00 ECRY 002 R! And AZOZ R! Iapkh 35 fifteen 1883.60 1493.93 1361,337 2037,139 14.00 58.00 1068.00 444.00 1413.00 755.00 2370.00 1485,00 6891,00 8193.00 ΤΝΡα ECRY 001 РГи АЗОЗ Р1иаг1х 35 fifteen 1174.74 1444.20 1119,633 1946,211 55.00 201.00 466.00 520.00 795.00 688.00 1720.00 1254.00 5415.00 7213.00 ECRY 002 Р1и АЗОЗ Р1иапх 35 fifteen 1545.40 1304.73 1159,490 1759,716 135.00 144.00 831.00 318.00 1203.00 824.00 1857.00 1171.00 5,354.00 7056.00

8Ό = standard deviation.

Mt, Mach = minimum, maximum.

C) 1 = first quartile.

C) 3 = third quartile.

N = number of subjects tested with available results.

When comparing the differences in the frequency of occurrence of antigen-specific SP4 T-lymphocytes between two vaccine groups according to the Wilcoxon test for each study, it is seen that all the p values for Exp1o-P1i-002 were less than 0.05 and were considered statistically significant (in favor of P1iA803) ( see table 28).

Table 28

Conclusion statistics: p values according to the criterion of the sum of Wilcoxon ranks between different groups on the 21st day for responses of antigen-specific CP4 T lymphocytes (kinetics)

Cytokine

Group

00Ϊ

002 όδϊ

002 oot

002

00Ϊ

002 oot

002

P value

0.9423

0,0300

0.8989

0,0361

0.8738

Ο? ΘΤ2Ϊ

0.9747

0.0216

0,9916

0,0514

P-value: Wilcoxon test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between 2 groups on day 21.

- 43 011419

Ιν.3.4. Kinetic analysis: immune response in case of Po81-Pro-vaccination.

The frequency of occurrence of antigen-specific C 1) 4 T-lymphocytes secreting in response for the time point (ροδΐ-рге) was summarized using descriptive statistics for each cytokine, and for each vaccine group, and for each study in the table. 29, for each study and for each vaccine group in the table. 31. The output statistics are presented in table. 30 and 32.

Table 29

Descriptive statistics of the difference between time points after vaccination (day 21) and before vaccination (day 0) for the response of specific C1) 4 T lymphocytes to vaccination (kinetics)

Cytokine Group Study N Avg 80 M1p O1 Media on az Mach All two Р1и АЗОЗ ECRY 001 ECRY 002 34 34 4837.56 1737.79 4476,129 1,450,177 -609.00 -2379,00 1888,00 936.00 3483.50 1664.50 8148.00 2743,00 19555.00 4669,00 P1iag1X EXTR 001 EXTR 002 fifteen fifteen 3103.53 1369.00 3,726,645 1,127,784 436.00 197.00 800.00 725.00 2283.00 869.00 3226,00 1808,00 15169.00 4676.00 S040Ts Р1и АЗОЗ ECRY 001 ECRY 002 33 33 4819.06 1694.73 4,489,788 1431,082 -718.00 2359.00 1799.00 921.00 3479,00 1659,00 8,288.00 2662,00 19,480.00 4575,00 Yayaph EXTR 001 EXTR 002 fifteen fifteen 3,090.00 1360.93 3,684,759 1,131,051 477.00 243.00 822.00 725.00 2189,00 860.00 3208,00 1687.00 15021.00 4743.00 ΙΡΝγ Р1и АЗОЗ ECRY 001 ECRY 002 33 33 3127.09 1167.85 2974,067 893,363 -453.00 -817.00 1325.00 633.00 1721.00 1207,00 5162.00 1803.00 13,296.00 2831.00 P1iaph EXTR 001 EXTR 002 fifteen fifteen 1660.13 851.87 1834,023 859,585 -84.00 148.00 480.00 294.00 1386.00 501.00 2284.00 1222.00 7120.00 3564,00 !! _ 2 Р1и АЗОЗ ECRY 001 ECRY 002 33 33 3950.18 1404.67 3878,538 1355,665 -358.00 2702.00 1309,00 719.00 2780,00 1341.00 6635,00 2109.00 16988,00 4,342.00 YaiaNh EXTR 001 EXTR 002 fifteen fifteen 2413.87 1117.80 3027.392 975.934 263.00 -258.00 674.00 575.00 1672.00 714.00 2425.00 1618.00 12273,00 3850.00 ΤΝΡα None Azozoz ECRY 001 ECRY 002 33 33 2627.36 1072.36 2574,458 1044,140 -825.00 -1816.00 862.00 447.00 1475.00 1000,00 4764,00 1752.00 9267.00 3310,00 P1iag1kh EXTR 001 EXTR 002 fifteen fifteen 1460.53 904.67 3115.174 974.958 -1586.00 32.00 251.00 338.00 813.00 752.00 1314.00 965.00 12,275.00 3,892.00

8Ό = standard deviation.

Mt, Mach = minimum, maximum.

C) 1 = first quartile.

C) 3 = third quartile.

N = number of subjects tested with available results.

When comparing the differences in the frequency of occurrence of antigen-specific C1-4 T cells between two studies according to the Wilcoxon test for each vaccine group, it can be seen that all p values for the P1iA803 group were less than 0.05 and were considered statistically significant (in favor of Exp1o-P1- 001) (see table 30).

Table 30

Conclusion statistics regarding the difference between time points after vaccination (day 21) and before vaccination (day 0): p values according to the Wilcoxon rank sum criterion between different studies on day 21 for antigen-specific responses C1) 4 T lymphocytes (kinetics)

Cytokine Group P value ALL FOR TWO Yaiazoz 0,0005 Yayaph 0.1300 C0401_ Yaiazoz 0,0007 Yayaph 0.0890 ΙΝΡγ Yaiazoz 0.0012 Yayaph 0,1103 P_2 Yaiazoz 0.0025 Yayaph 0.1409 ΤΝΡα Yaiazoz 0,0327 Yayaph 0.6936

- 44 011419

P-value: Wilcoxon test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between the two groups on day 21.

Table 31 Descriptive statistics of the difference between time points after vaccination (day 21) and before vaccination (day 0) for the response of specific CE4 T lymphocytes to vaccination (kinetics)

ECRY 002

ECRE

001

ECRY 001

ECRY 001

ECRE

002

ECRE

002

ECRY 002

ECRY 001

Explored bath

ECRE

002

ECRY 001

Group

Avg

M1p

Median

Neither A503 Niaph Ne A803 Niaph Ne A803 NiaNh Ai A603 Niag1h Ty

A503 Yaiag1x

Ni A503

Niap Ni A803 Niap’x

None A503 Niahp None A803 NiaNh Nee A603 Niahp

4476,129

-609.00

1888,00

3483.50

8148.00

19555.00

D5

3103.53

1737.79

1369.00

4819.06

3,090.00

1694.73

1360.93

3127.09

1660.13

1187.85

851.87

3950.18

1117.80

2627.36

1460.53

1072.36

904.67

3,726,645

1,450,177

1127,784

4,489,788

3684,759

1431,082

1131,051

2974,067

1834,023

893,363

859,585

3878,538

3027,392

1355,665

975,934

2574,458

3115,174

1044,140

974,958

800.00

936.00

2283.00

1664.50

3226,00

2743,00

15169.00

4669.00 _197.00

-718.00

725.00

1799.00

869.00

3479,00

1808,00

8,288.00

4676,00

19,480.00

477.00

-2359,00

2189,00

1659,00

3208,00

2662,00

15021.00 4575.00

243.00 -453.00

-84.00 -817.00

148.00

-358.00

263.00 -2702.00

-258.00

-825.00

-1586.00

-1816.00

32.00

725.00

1325.00

860.00

1721.00

1687.00

5162.00

4743,00

13,296.00

480.00

633.00

1386.00

1207,00

2284.00

1803.00

7120.00

2831.00

294.00

1309,00

501.00

2780,00

1222.00

6635,00

3564,00

16988,00

674.00

719.00

575.00

862.00

251.00

447.00

338.00

1672.00

1341.00

2425.00

2109.00

12273,00

4,342.00

714.00

1475.00

1618.00

4764,00

3850.00

9267.00

813.00

1000,00

1314.00

1752.00

12,275.00

3310,00

752.00

8Ώ = standard deviation.

Mt, Mach = minimum, maximum.

= first quartile.

(.33 = third quartile.

N = number of subjects tested with available results.

965.00

3892,00

When comparing the differences in the frequency of occurrence of antigen-specific C1-4 T cells between two vaccine groups according to the Wilcoxon test for each study, it is clear that p is less than 0.05 only for Exp1o-G1i-001 and it was considered statistically significant (in favor of G1iA803 ) (see table 32).

Table 32

Conclusion statistics: p values according to the criterion for the sum of Wilcoxon ranks between different groups on the 21st day for antigen-specific responses C1) 4 T-lymphocytes (kinetics)

Cytokine Group P value ALL FOR TWO ΕχρΙο None 001 0.0827 ΕχρΙο None 002 0,0992 СО401- ΕχρΙο None 001 0.0931 ΕχρΙο None 002 0,1391 ! ΝΡγ ΕχρΙο None 001 0,0543 ΕχρΙο None 002 0.1068 11_2 ΕχρΙο None 001 0.0847 ΕχρΙο None 002 0.2254 ΤΝΡα ΕχρΙο None 001 0,0375 ΕχρΙο None 002 0,2009

- 45 011419

P-value: Wilcoxon test (non-parametric technique) for testing the detected difference (Wilcoxon rank sum test) between the two groups on day 21.

1U.4. W-captions.

The results are shown in FIG. 9 and tab. 33-36.

Table 33 Geometric mean titers (SMT) and seropositivity levels of anti-W-titers (SMT calculated for vaccinated subjects)

Antibody Group Schedule N 3+ % 95% CI SMT 95% CI and. υι ίί υι Α / Νβνν Ca1 Piaph PPE eighteen 17 94.4 72.6 99.9 63.5 38.1 105.9 ΡΙ (ϋ21) eighteen eighteen one hundred 81.5 one hundred 131.9 77.1 225.6 Pyazosis PPE 40 39 97.5 86.8 99.9 70.3 50,5 97.7 ΡΙ (ϋ21) 40 40 one hundred 91.3 one hundred 218.6 158.2 302.0 A / Eifap Piaph PPE eighteen eighteen one hundred 81.5 one hundred 95.0 51.0 176.9 ΡΙ (ϋ21) eighteen eighteen one hundred 81.5 one hundred 498.3 272.1 912.7 Pyazosis RVE 40 40 one hundred 91.3 one hundred 94.3 71,4 124.6 ΡΙ (ϋ21) 40 40 one hundred 91.3 one hundred 735.1 564.4 957.5 B / b On1 Piaph RVE eighteen sixteen 88.9 65.3 98.6 23.3 15,2 35.8 ΡΙ (ϋ21) eighteen 17 94.4 72.6 99.9 139.8 64.0 305.0 Pyazosis PPE 40 38 95.0 83.1 99,4 58.6 43.9 78.1 ΡΙ (ϋ21) 40 40 one hundred 91.3 one hundred 364.4 269.7 492.4

PBE = before vaccination.

^ (021) = 21st day after vaccination.

95% C1, b, and b = 95% confidence interval, lower and upper limits. 8+ = number of seropositive subjects.

Table 34

Anti-III titer conversion factor (all vaccinated subjects)

Group Α / Νθνν Ca! Ec1ota A / Rirap B / 5NapdNa1 N QMS [95% CI] N QMS [95% CI] N (ZMK [95% CI] Piaph eighteen 2.1 [1.4: 3.2] eighteen 5.2 [h, o; 9, h] eighteen 6.0 EZ, 5; 10.2] Pyazosis 40 3.1 [2.4; 4.0] 40 7.8 [5.6; 10.9] 40 6.2 [4.7; 8.2]

Ν = total number of subjects.

SMV = geometric mean ratio (antilogarithm of the average logarithm of the ratio of titers on the 21st day / 0th day).

95% CI = 95% confidence interval.

Table 35 Seroprotection levels of anti-W-titers (all vaccinated subjects)

Antibody Group Schedule N η > = % 40 95% CI Α / Νθνν Ca1e0ogpa Piaph PPE ΡΙ (ϋ21) eighteen eighteen 14 sixteen 77.8 88.9 52,4 65.3 93.6 98.6 Pyazosis PPE ΡΙ (ϋ21) 40 40 32 39 80 97.5 64,4 86.8 90.9 99.9 A / Rirap Piaph PPE ΡΙ (ϋ21) eighteen eighteen 14 eighteen 77.8 one hundred 52.4 81.5 93.6 one hundred Pyazosis PPE ΡΙ (ϋ21) 40 40 36 40 90 one hundred 76.3 91.2 97.2 one hundred B / 8 Piaph PPE ΡΙ (ϋ21) eighteen eighteen 6 14 33.3 77.8 13.3 52.4 59.0 93.6 Pyazosis PPE ΡΙ (ϋ21) 40 40 34 40 85 one hundred 70.2 91.2 94.3 one hundred

PBE = before vaccination.

^ (021) = 21st day after vaccination.

Ν = number of subjects with available results.

n = number of subjects with captions within a specific range. % = percentage of subjects with captions within a specific range.

- 46 011419

Table 36

Seroconversion levels on day 21 after vaccination (a multiple increase of 4) (all vaccinated subjects)

Antibody Vaccine group N η Resp % underders 95% IX DI Α / Νθνν Ca1ec1op1a Piaph eighteen 3 16.7 3.6 41.5 NIABOZ 40 nineteen 47.5 31.5 63.9 A / Ecrap Niaph eighteen thirteen 72,2 46.5 90.3 NIAZOZ 40 34 85.0 70,2 94.3 W / Zpappda! Niaph eighteen 12 66.7 41.0 86.7 NIAZOZ 40 31 77.5 61.5 89.2

N = number of subjects with available results both before and after vaccination.

and = number of responders.

% = share of responders (IZh100).

95% CI = strictly 95% confidence interval; 14, = lower limit, CT = upper limit.

GU. 5. General conclusions.

Based on this clinical study, the Ni-A803 adjuvant vaccine is superior to the equivalent non-adjuvant Niaph vaccine in the frequency of occurrence of influenza-specific C1) 4 T cells, as well as in the persistence of the immune response caused by the first Ni-A803 vaccination (initial vaccination in Exp1o -P1i-001) until the time of Ώ0 revaccination study (Exp1o-P1i-002, i.e. after +/- 1 year). In addition, this response is able to recognize the drifting strains of the influenza virus present in the new vaccine and to recognize the 2004 influenza vaccine strains.

In contrast to first year vaccination, after revaccination, individuals pre-vaccinated with adjuvant Niap ™ showed increased H-titer reactivity compared with vaccines with non-adjuvant Niap ™. There is a noticeable tendency to a 1.5-2-fold increase in the Sh-titer against strains NSh1 and No. N2 and the demonstrated statistical increase in the Sh-titer against strain B.

Example V. Preclinical evaluation of adjuvant and non-adjuvant ferret influenza vaccines.

First study. The effectiveness of the new compositions A803 and A803 + MPB.

V. 1. Rationale and objectives.

Influenza in the ferret model closely mimics the flu in humans, both in terms of susceptibility to infection and the clinical response.

The ferret is very sensitive to infection with both the influenza A and B viruses without prior adaptation of the viral strains. Consequently, this provides an excellent model system for the protection studies provided by the administered influenza vaccines.

This study examined the efficacy of various trivalent split vaccines, adjuvanted or non-adjuvanted, to alleviate disease symptoms (body temperature) and viral shedding in nasal secrets of ferrets that were challenged with homologous strains.

The objective of this experiment was to demonstrate the efficacy of an adjuvanted influenza vaccine compared to a simple (non-adjuvanted) vaccine.

The following endpoints were determined:

1) primary endpoint: reduction of viral shedding in nasal washes after homologous control infection;

2) secondary endpoints: analysis of the humoral response by means of an SA (indirect hemagglutination reaction) and monitoring of the temperature near the point of priming and control infection.

D.2. Experiment plan.

D.2.1. Treatment / group (tab. 37).

Female ferrets (Mi8ίe1a pHopiz Σηγο) (6 ferrets / group) at the age of 14-20 weeks were received from \ P8A ¥ Soizi Naisu (Natrzyg, IR). Ferrets were primed on the 0th day with the heterosubtypic strain NSh1 L / 81osk11o1t / 24/90 (4 1od of TSGO ₅₀ / ml). On the 21st day, the ferrets were given an intramuscular injection of the full human dose (BUT) (500 μg of the vaccine dose, 15 μg of HA / strain) of the combination NSh1 A / \ e \ with Ca1eyosha / 20/99, No. N2 A / Raiata / 2007 / 99 and B / 811a11ds1o11d / 7/97. Then the control infection of the ferrets was carried out on the 41st day via the intranasal route with the homotypic strain No. N2 A / Raiata / 2007/99 (4.51 1od TSGO50 / ml).

- 47 011419

Table 37

Group Antigen (s) + dosage Composition + Dosage Notes (mode / path / infection) Other treatments one Trivalent simple Full nude: 15 mcg HA / strain in / m; 21st day Priming Η1Ν1 (A / 8UskNo1t / 24/90) on day 0 2 Trivalent A803 Full Nude: 15 mcg HA / strain in / m; 21st day Priming Η1Ν1 (A / 81oskobo1t / 24/90) on the 0th day 3 Trivalent Α803 + ΜΡΙ- Full Ηϋ: 15 mcg HA / strain in / m; 21st day Priming Η1Ν1 (A / 81oscpo1t / 24/90) on the 0th day 4 PB8 Full Ηϋ: 15 mcg HA / strain in / m; 21st day Priming Η1Ν1 (A / 81osk1to1t / 24/90) on the 0th day

ν.2.2. Preparation of vaccine compositions.

Composition 1. Trivalent simple (non-adjuvanted) composition (500 μl).

Tenfold concentrated PB8 (pH 7.4, based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and UE8 (in quantities taking into account the detergents present in the strains), are added to water for injection. The amounts of detergents reach the following values: 750 μg Tween 80, 110 μg Triton X-100 and 100 μg UE8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and 17.5 μg of strain B are added, each time with 10 minutes stirring between additions. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

Composition 2. Trivalent cleaved anti-influenza with adjuvant Α803 (500 μl).

Tenfold concentrated PB8 (pH 7.4, based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and UE8 (in quantities taking into account the detergents present in the strains), are added to water for injection. The amounts of detergents reach the following values: 750 μg Tween 80, 110 μg Triton X-100 and 100 μg UE8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and 17.5 μg of strain B are added, each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion (prepared as explained in Example A.1.) Is added. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

Composition 3. Trivalent cleaved anti-influenza with adjuvant A803 + MPB.

Tenfold concentrated PB8 (pH 7.4, based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and UE8 (in quantities taking into account the detergents present in the strains), are added to water for injection. The amounts of detergents reach the following values: 750 μg Tween 80, 110 μg Triton X-100 and 100 μg UE8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and 17.5 μg of strain B are added, each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion (prepared as explained in Example A.1.) Is added. The mixture was again stirred for 15 minutes immediately before adding 25 μg MPB from the suspension prepared as described in detail in Example A.3.1. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

Note: ten times concentrated PB8 is added to each composition to achieve isotonicity and a single concentration in the final volume. The volume of H ₂ About calculated to achieve a given volume.

ν.2.3. Reading results (table. 38).

Table 38

Reading result Time point Sample Type Ι / Ρ Analysis Method Viral shedding 1 day before and within 7 days after priming. 1 day before and within 5 days after infection control Nasal swabs Ιη Titration Monitoring T ° 1 day before and within 3 days after priming. 2 days before and within 3 days after infection control Peritoneal implant Ιη Telemetry ΙΗΑ Before, after priming, after immunization, after challenge Serum Ιπ ΙΗΑ

S = individually / Po = pool.

- 48 011419

ν.3. Results.

A schematic representation of the results is shown in FIG. 10 and 11.

ν.3.1. Temperature monitoring.

Individual temperature was recorded using sensors and telemetry recording (in accordance with the procedure described in detail in A.2.2.). All implants were checked and updated and a new calibration was performed using Ό8Ι before placement in the intraperitoneal cavity. At the time of these measurements, all animals were individually housed in separate cages.

The temperature was recorded every 15 min, starting from 3 days before the control infection and up to 5 days after the control infection, and the average value was calculated in the middle of the day. The results of the change in body temperature from the baseline to the baseline are shown in FIG. 10 A (results from -1 to +3 days are shown) and 10B (results from -2 to +3 days are shown).

After challenge, a peak in body temperature was observed only after immunization with the trivalent simple split composition or RVB. No peak was observed after immunization with the trivalent split composition with AB03 or AB03 + MP adjuvant.

ν.3.2. Viral shedding (Fig. 11).

Titration of viruses in nasal washes was performed on 6 animals per group.

Nasal swabs were obtained by introducing 5 ml of RVB into both nostrils of awake animals. The inoculum was collected in a Petri dish and placed in sample containers at -80 ° C (dry ice).

All nasal samples were first filtered under sterile conditions through Brsh X (Co81ag) filters to remove any bacterial contamination. 50 μl of serial ten-fold dilutions of nasal washings were transferred to microtiter plates containing 50 μl of medium (10 wells / dilution). Then, 100 μl of MOSCOW cells (2.4 × ^{10 5} cells / ml) were added to each well and incubated at 35 ° C until the cells reached confluence for control cells, for example, for 5–7 days. After 6-7 days of incubation, the culture medium is carefully removed, 100 μl of 1/20 νδΤ-1-containing medium are added and incubated for another 18 hours.

The intensity of the yellow formazan dye resulting from the restoration of ν8Τ-1 by viable cells is proportional to the number of viable cells present in the well by the end of the virus titration analysis and is quantified by measuring the absorbance at the corresponding wavelength (450 nm). The reference point is defined as the average OE value for uninfected control cells - 0.3 OE (0.3 OE correspond to +/- 3 BO (standard deviation) OE for uninfected control cells). A positive score is determined when the TOE is below the reference point, and, conversely, a negative score is determined when the TOE is above the reference point. Viral shedding titers were determined according to Kei apy Miepsy and expressed as 1ode TCU ₅₀ / ml.

Weaker viral shedding was observed after challenge with the trivalent split composition with the AB03 or AB03 + MPI adjuvant compared to the trivalent simple split composition or the RBB. The protective effect was slightly better with AB03 compared with AB03 + MPI (see day 2 after challenge). Statistical significance could not be determined due to the small number of animals in the group.

ν.3.3. Conclusion from the experiment.

Higher humoral responses (тит-titers) were observed with the trivalent split composition with the AB03 or AB03 + MPI adjuvant compared to the trivalent simple split composition for all three strains (at least two-fold for two of the three strains, i.e., for strains Η3Ν2 and B).

Compositions with AB03 and AB03 + MPR have demonstrated additional benefits in terms of the effectiveness of protection on ferrets (lower viral shedding and temperature) (FIGS. 10 and 11).

After infection control, no increase in the humoral response was observed after immunization with the trivalent split composition with the adjuvant AB03 or AB03 + MPB.

The second study. Study of heterotypic control infection on ferrets: demonstration of the effectiveness of the tested new composition.

ν.4. Justification and objectives.

This study examined the efficacy of various trivalent split vaccines, adjuvanted or non-adjuvanted, on their ability to reduce disease symptoms (body temperature) and their effect on viral shedding in nasal secretions in immunized ferrets after a heterologous challenge.

ν.5. Experiment plan.

Female ferrets (Miye1a ri1ogsh8 Digo) (6 ferrets / group) at the age of 14-20 weeks were received from MKAU Sopkikapsu IR). Four groups tested

P1iapkh, trivalent split-AB03, trivalent split-AB03 + MPB, * RVB.

Ferrets were primiered on the 0th day with the heterosubtypic strain Η1Ν1 A / B1oskyo1t / 24/90 (4 1 od TSDSO ₅₀ / ml). On the 21st day, ferrets were administered to a ferret through an intramuscular injection

- 49 011419 dose (500 μg of the vaccine dose, 15 μg of HA / strain) of the combination Η1Ν1 Λ / No. \ ν Ca1syosha / 20/99, Η3Ν2 A / Rapata / 2007/99 and B / 8apdbopd / 7/97 (17.5 mcg ON). Then the control ferrets were infected on the 43rd day via the intranasal route with the heterosubtypic strain Η3Ν2 A / ^ wattsd / 3/2003 (4.51 1od TSN), / ml).

ν.6. Results.

A schematic representation of the results is shown in FIG. 12 and 13. ν.6.1. Temperature monitoring.

Individual temperature was recorded using sensors and telemetry recording. All implants were checked and updated and a new calibration was performed using Ό8Ι before placement in the intraperitoneal cavity. At the time of these measurements, all animals were individually housed in separate cages.

The results (Fig. 12) show that near the point of priming observed greater variability from one group to another. The baseline looked higher before priming than after priming;

despite the great variability in body temperature, the peak was observed only after challenge in ferrets immunized with PB8 (6/6 ferrets), a trivalent simple split composition (5/6 ferrets) and a trivalent split composition with A803 adjuvant (2/6 ferrets ) No peak was observed after immunization with the trivalent split composition with adjuvant A803 + MPB (0/6 ferrets);

A803-Composition seems to be less effective than A803 + MPB against heterologous strains in preventing fever. The authors cannot conclude that it is possible that the difference between adjuvants is due to different levels of antibodies before the challenge.

ν.6.2. Viral shedding (Fig. 13).

Nasal swabs were obtained by introducing 5 ml of PB8 into both nostrils of awake animals. The inoculum was collected in a Petri dish and placed in sample containers at -80 ° C (dry ice).

All nasal samples were first filtered under sterile conditions through 8px X filters (Co §1at) to remove any bacterial contamination. 50 μl of serial ten-fold dilutions of nasal washings were transferred to microtiter plates containing 50 μl of medium (10 wells / dilution). Then, 100 μl of MOSCOW cells (2.4 x 10 ⁵ cells / ml) were added to each well and incubated at 35 ° C until the cells reached confluence for control cells, for example, for 5-7 days. After 6-7 days of incubation, the culture medium is carefully removed, 100 μl of 1/20 ^ 8T-1-containing medium are added and incubated for another 18 hours.

The intensity of the yellow formazan dye resulting from the restoration of \ U8T-1 by viable cells is proportional to the number of viable cells present in the well by the end of the virus titration analysis and is quantified by measuring the absorbance at the corresponding wavelength (450 nm). The reference point is defined as the average value ΟΏ for uninfected control cells - 0.3 ΟΌ (0.3 ΟΌ corresponds to +/- 3 8Ό (standard deviation) ΟΌ for uninfected control cells). A positive score is determined when ΟΌ is below the reference point, and, conversely, a negative score is determined when ΟΌ is above the reference point. Viral shedding titers were determined according to Kssb apb MispSb and expressed as 1ode TSGO ₅₀ / ml.

Viral shedding after priming

Viral shedding was measured for 12 ferrets, starting from 1 day before priming and on the 7th day after priming. Results are expressed as a pool.

Viral clearance was observed on the 7th day after priming in all ferrets.

Viral shedding after challenge

Viral shedding was measured for 6 ferrets / group, starting from 1 day before the challenge and 7 days after the challenge.

2 days after the challenge, lower statistically significant viral titers were observed in ferrets immunized with the trivalent split composition with adjuvant A803 and A803 + MPB compared with ferrets immunized with the trivalent simple split composition and PB8 (difference 1.25 / 1, 22 logarithms and 1.67 / 1.64 logarithms for groups with adjuvant A803 / Ag03 + MPB compared with a simple vaccine, respectively).

On the 50th day, viruses were not detected in nasal swabs.

ν.6.3. Hemagglutination Inhibition Test (ΗΙ-titers) (Fig. 14A and B).

Serum samples were collected on the 1st day before priming, the 21st day after priming, the 2nd day after immunization, and the 14th day after challenge.

Anti-hemagglutinin antibody titers of the Η3Ν2 influenza virus (vaccine strain and strain for control infection) were determined using a hemagglutination inhibition test (ΗΙ). The principle of the теста test is based on the ability of specific anti-influenza antibodies to inhibit the hemogglutination of red blood cells (BBC) of the chicken with hemagglutinin (HA) of the influenza virus. Serum samples were initially treated with a 25% solution of neuraminidase (CFU) and heat inactivated to remove non-specific inhibitors. After pretreatment, double serum dilutions

- 50 011419 were incubated with 4 units. hemagglutination of each strain of influenza virus. Then chicken red blood cells were added and inhibition of agglutination was evaluated. The titers were expressed as the reciprocal of the highest dilution of serum at which complete inhibition of hemagglutination was observed. Since the first dilution of serum was 1:10, an undetectable level was evaluated as a titer of 5.

results

The results are shown in FIG. 14A and 14B. After immunization with Η3Ν2 A / Rapata, higher humoral responses (тит-titers) were observed in ferrets immunized with a trivalent split vaccine with adjuvant A803 or A803 + MPB compared with the humoral response observed after immunization of ferrets with a non-adjuvant (non-adjuvant) vaccine (Niaph ™).

Similar ΗΙ-titers were observed in ferrets immunized with Η3Ν2 A / Rapata with adjuvant A803 or A803 + MPB.

Cross-reactive тит-titers to the heterologous strain ALUuott Η3Ν2 were observed only after immunization with the vaccine containing strain A / Rapata Η3Ν2 with adjuvant A803 or A803 + MPB (they were not observed after immunization with the trivalent simple split vaccine).

An increase in ALUuotd-specific тит-titers was observed in ferrets immunized with the heterologous strain A / Rapata Η3Ν2 and subjected to control infection with AЖuottd Η3Ν2. As expected, and in contrast to homologous control infection, heterologous control infection led to an increase in A / Rapata-specific тит-titers in ferrets immunized with A / Rapata Η3Ν2 with adjuvant A803 and A803 + MPI.

ν.6.4. Conclusion on this experiment.

As expected, an increase in anti-H3N2 тит titers was observed after heterologous control infection compared with the situation after homologous control infection (no increase).

However, after a heterologous and homologous challenge, a similar protection was observed (viral shedding).

Example νΙ. Preclinical evaluation of adjuvant and non-adjuvant anti-influenza vaccines in primed C57Y / 6 mice.

νΙ. 1. Experiment plan and task.

Significantly higher CE4 T-cell responses were observed in a clinical study of Exρ1o-Ε1i-001 (see Example ΙΙΙ) for trivalent Ε1i-split-Α§03 compared to simple Niap.x (non-adjuvant). No difference was found between the two groups for both CE8 T-cell and humoral responses.

The task was to select readings of CMI-induced responses in mice similar to those observed in humans. In particular, the objective was to demonstrate higher CMI responses in mice as a result of using split-Ag03 or split-A803 + MPB compared to a simple split composition.

νΙ.1.1. Processing / group.

Female C57Y / 6 mice (15 mice / group) aged 6-8 weeks were received from Η-ιΊ-ιι Netherlands. The tested groups were trivalent split simple, trivalent split-Ag03, trivalent split-A803 + MPB,

PB8.

Mice were primed on the 0th day with heterosubtypic strains (5 μg of HA-containing whole inactivated Η1Ν1 A / 1appe8bigd / 82/96, Η3Ν2 A / 8ubpeu / 5/97, B / W-cnl / M). Ηι On the 28th day, mice were injected by intramuscular injection of 1.5 μg of a HA-containing trivalent split composition (A / No \ in Ca1ebosha / 20/99, A / Rapata / 2007/99, B / §yapdbopd / 7/97) , simple or adjuvant (groups see below).

νΙ.1.2. Preparation of vaccine compositions.

Tenfold concentrated PB8 is added to each composition to achieve isotonicity and a single concentration in the final volume. To achieve a given volume, a volume of Η ₂ Ο is calculated.

Trivalent simple (non-adjuvant) split composition

Composition 1 (for 500 μl). Tenfold concentrated PB8 (pH 7.4, based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and νΈδ (in quantities, taking into account the detergents present in the strains), are added to water for injection. The amounts of detergents reach the following values: 750 μg Tween 80, 110 μg Triton X-100 and 100 μg νΈδ per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

Trivalent split composition supplemented with A803 adjuvant in the form of an oil-in-water emulsion

Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and νΈδ (in quantities taking into account the detergents present

- 51 011419 in strains), added to water for injection. The amounts of detergents reach the following values: 750 μg Tween 80, 110 μg Triton X-100 and 100 μg UE8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion (prepared as explained in Example A.1) is added. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

Trivalent split composition with adjuvant Λ803 + ΜΡΕ

Tenfold concentrated ΡΒ8 (pH 7.4 based on a single concentration), as well as a mixture containing Tween 80, Triton X-100, and UE8 (in quantities, taking into account the detergents present in the strains), are added to water for injection. The amounts of detergents reach the following values: 750 μg Tween 80, 110 μg Triton X-100 and 100 μg UE8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion (prepared as explained in Example A.1) is added. The mixture is again stirred for 15 minutes immediately before adding 25 μg. ΜΡΒ The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, if not administered immediately.

UT1.3. Reading results.

Analysis of СΜI ЦС8: СО4 / СЭ8, staining for ГB-2 / ΙΡΝγ).

МиC was collected from the primed mice 7 days after immunization. They were tested in pools / group.

U12. Results.

Conditions demonstrating a higher incidence of ί.Ό4 and CE8 + T cells, as well as a lower background, were determined using C57BL6 primed mice and whole inactivated virus (1 μg / ml) as antigen for re-stimulation. The results are shown in FIG. 15 (ί.Ό4 T-cell responses) and in FIG. 16 (SE8 T cell response).

Under these conditions, it was possible to induce higher ί.Ό4 T-cell responses for split-A803 compared to a simple split-composition, as was observed in humans;

higher ί.Ό4 T-cell responses for split-A803 + ΜΡ ^ as compared to a simple split-composition;

similar CE8 T cell responses for a simple split composition and split-A803, as observed in humans;

a tendency to higher ί.Ό8 T-cell responses for A803 + ΜΡ ^ compared to split A803 or a simple split composition.

UE example. Preclinical evaluation of adjuvant and non-adjuvant anti-influenza cleaved and subunit vaccines in C57BE6 mice primed with heterologous strains.

UP.1. Experiment plan and task.

Significantly higher P4-T cell responses were observed in the clinical study Exp1o-P1i-001 (see Example ΙΙΙ) for trivalent P1i-split-A803 compared to simple P1iapch (non-adjuvant). No difference was found between the two groups for both CE8 T-cell and humoral responses.

Using C57BE6 mice primed with heterologous strains, an animal model was developed that reproduces the similar immune profiles observed in humans. For Κ.'8 (intracellular staining of cytokines), repeated stimulation was carried out using inactivated whole virus.

The task was to compare SM! the response induced by the commercially available C1axo8bK11 split vaccine (P1iapx ™) compared to the subunit vaccine (P1iab ™ vaccine from Sygoi), as well as the CI response obtained using these vaccines with adjuvant A803 or A803 + ΜΡ ^ or another adjuvant as an oil-in-water (Ον) emulsion.

UP.1.1. Processing / group.

Female C57BE6 mice (24 mice / group) aged 6-8 weeks were received from Magk-sh Chogh !, Netherlands. Mice were primed intranasally on day 0 with heterosubtypic strains (5 μg of HA-containing whole inactivated with formaldehyde Η1Ν1 ADokiiiekigd ^ W, Η3Ν2 A / 8ubieu / 5/97, B / Chagyu / TICH). On the 29th day, mice were injected with mice by intramuscular injection of 1.5 μg of HA-containing trivalent split composition (A / Le ^ Ca1ebosha / 20/99, Azhuot1id / 3/2003, B / Laidki / 10/2003), simple or adjuvant (see groups in table 39, below).

- 52 011419

Table 39

Gr.

Antigen / Composition

Other treatment

one Trivalent split * / simple (non-adjuvant) = Piap ™ 0-day heterologous priming 2 Trivalent split * / O \ L / 0-day heterologous priming 3 Trivalent split * / AZOZ 0-day heterologous priming 4 Trivalent split * / AZOZ + MRI (2.5 μg per dose) 0-day heterologous priming 5 Spdiags! (= Piad ™) = subunit in an oil-in-water emulsion 0-day heterologous priming 6 Addpra! ™ (subunit) / AZOZ 0-day heterologous priming 7 Addpra! ™ (subunit) / AZOZ + MRI (2.5 mcg per dose) 0-day heterologous priming 8 Addpra! ™ (subunit) / Ονν ** 0-day heterologous priming nine Addpra! ™ (subunit) 0-day heterologous priming 10 RVZ 0-day heterologous priming

* P1iapkh ™.

** The coolant was prepared as described in the section below.

νΙΙ.1.2. Preparation of vaccine compositions.

Coolant preparation

An oil-in-water emulsion labeled with coolant (from English o11-1n- ^ a! Eg) is prepared according to the recipe published in the leaflet with the instructions attached to the R1iAb vaccine from Syndrome Vepp.

Water for injection, 36.67 mg of citric acid and 627.4 mg of No. citrate-21 PO were mixed and the volume was adjusted to 200 ml. 470 mg of Tween 80 is mixed with 94.47 ml of this buffer and this mixture is designated as solution A. The oil mixture is prepared by stirring 3.9 g of squalene and 470 mg of 8rap 85 on a magnetic stirrer. Next, solution A and the final volume obtained are added to the oil mixture. is 100 ml. Then the mixture was first passed through a needle 18Oh a 1 _^1/2, and thereafter placed in a microfluidizer M1108 (from MyugoPshShsz) as two specimens for reducing the size of oil droplets. When a particle size of approximately 150 nm is obtained for each sample, the two samples are combined and filtered on a 0.2 μm filter. An average ζ value of 143 nm with a polydispersity of 0.10 is obtained for the combined sample at time T0, and after 4 months of storage at 4 ° C, 145 nm with a polydispersity of 0.06. This size is obtained using Ζеΐа8 ^ ζе ^ 3000Η8 (from Macet) in the following specifications:

laser wavelength: 532 nm (2е1-3000Н8), laser power: 50 mW (2е1-3000Н8), scattered light detected at 90 ° (2е1-3000Н8), temperature: 25 ° С, duration: programmed automated determination, quantity: 3 consecutive measurements, average diameter ζ: according to cumulative analysis.

Composition for group 1 (for 1 ml)

Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and νΕ8 (in quantities taking into account the detergents present in the strains) are added to water for injection to achieve the final concentration 375 μg / ml Tween 80, 55 μg / ml Triton X-100 and 50 μg / ml νΕ8. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. The composition is stirred for 15 minutes and stored at 4 ^° C, unless administered immediately.

Composition for group 2 (for 1 ml)

Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and νΕ8 (in quantities taking into account the detergents present in the strains), are added to water for injection to achieve the final concentration 375 μg / ml Tween 80, 55 μg / ml Triton X-100 and 50 μg / ml νΕ8. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of coolant emulsion are added. The composition is stirred for 15 minutes and stored at 4 ° C if not administered immediately.

Composition for group 3 (for 1 ml)

Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as

- 53 011419 a mixture containing Tween 80, Triton X-100 and UE8 (in quantities, taking into account the detergents present in the strains), is added to water for injection until a final concentration of 375 μg / ml Tween 80, 55 μg / ml Triton X- 100 and 50 μg / ml UE8. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion is added. The composition is stirred for 15 minutes and stored at 4 ° C if not administered immediately.

Composition for group 4 (for 1 ml)

Tenfold concentrated PB8 (pH 7.4, based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and UE8 (in quantities taking into account the detergents present in the strains), are added to water for injection until the final concentration is reached 375 μg / ml Tween 80, 55 μg / ml Triton X-100 and 50 μg / ml UE8. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion is added. The mixture was re-mixed for 15 minutes immediately before the addition of 25 μl of ShRK. The composition was mixed for 15 minutes and stored at 4 ° C, if not administered immediately.

Composition for group 5 (for 1 ml)

The same volumes of PB8 and the AIAi ™ / Spryagi ™ vaccine (commercially available vaccine) are mixed. The composition is stirred for 15 minutes and stored at 4 ° C if not administered immediately.

Composition for group 6 (for 1 ml)

250 μl of PB8-mod, pH 7.4, is added to a dose of 500 μl of Addpra1 ™ (a commercially available vaccine). After 15 minutes of stirring, 250 μl of 8B62 (prepared according to the procedure described in detail in the section Preparation on an enlarged scale) is added. The composition is stirred for 15 minutes and stored at 4 ° C if not administered immediately.

Composition for group 7 (for 1 ml)

PB8-mod, pH 7.4 (to achieve a final volume of 1 ml), is added to a dose of 500 μl of AddpraG ™ (a commercially available vaccine). After 15 minutes of stirring, 250 μl of 8B62 (prepared according to the procedure described in detail in the section Preparation on an enlarged scale) is added. Then add 25 μg MPI. The composition is stirred for 15 minutes and stored at 4 ° C if not administered immediately.

Composition for group 8 (for 1 ml)

250 μl of PB8-mod, pH 7.4, are added to a dose of 500 μl of Ag§l1 After 15 minutes of stirring, add 250 μl of OA prepared as for group 2, the composition is stirred for 15 minutes and stored at 4 ° C, if not administered immediately.

Composition for group 9 (for 1 ml)

The same volumes of PB8 modes, pH 7.4, and Addpra1 are mixed. The composition is stirred for 15 minutes and stored at 4 ° C if not administered immediately.

UP.1.3. Reading results (table. 40).

CMI CS8): 7 days after immunization.

Sha / neutralization analysis: 21 days after immunization.

Table 40

Reading result Time point Sample Type Ιη / Ρο Analysis Method 1C8 (СО4, СО8, ΙΙ - 2, ΙΓΝ-γ) 35th day RVI Ro PAC8 analysis Humoral answer 14th day, 44th day Serum Ιπ ΙΗΑ neutralization

And = individually / Po = pool.

Analysis of CMI TsS8: SP4 / SE8; staining for W ^ LH ^ gamma)

PBMCs were collected from 24 mice / group 7 days after immunization and tested in pools / group. UP.2. Results.

UP.2.1. Humoral immunity.

Hemagglutination inhibition activity against three vaccine strains was detected in serum samples from 24 animals per group on the 14th day after intranasal heterologous priming and on the 16th day after immunization.

For three strains and for all groups, an increase in W titers was observed after immunization.

For the same adjuvant and for three strains, similar S-titers were induced by subunit vaccine and split vaccine.

Similar W titers were observed for Oiai in comparison with Addpra1 OA for three strains. No differences were observed between IIaph and Addpra1 for strains Η1Ν1 and B.

For the three strains, higher statistically significant S titers were observed when the A803 adjuvant with or without MPB was present in the II vaccine (split or subunit), compared

- 54 011419 with a simple AI vaccine.

S-titers were statistically significantly higher for a P1i vaccine (split or subunit) with an OA adjuvant compared to a simple P1i vaccine for strain A / Auot only.

UP.2.2. Cell-mediated immune response (Ι08 on the 7th day after immunization).

ί.Ό4 T-cell responses - FIG. 17, the upper part

RVMS from 24 mice per group were collected on the 7th day after immunization and tested in 1 pool / group. Inactivated trivalent whole viruses (1 μg / ml) were used as antigen for repeated stimulation. The results are shown in FIG. 17, the upper part.

As for the whole CE1 + P4 virus-specific CE4 + T cells expressing ΙΕ-2, ΙΕΝ-γ or both cytokines (Fig. 17, upper part):

1) adjuvants from O8K (O1axo8t1DK1ts) showed the same tendency that was observed earlier (example Y!): A803 + MPB was better than A803, which, in turn, was superior to the results obtained with a simple vaccine. This trend has been observed for both split and subunit vaccines;

2) whatever the composition (simple, A803- or A803 + MRB-), the split vaccine induced higher ί.Ό4 + T-cell responses than the subunit vaccine;

3) P1iab (subunit + OA-type oil-in-water emulsion - see Preparation section), apparently, induces the same frequency of occurrence as simple P1iapkh;

4) trivalent split / A803 or trivalent split / A803 + MPI composition induced higher ί.Ό4 + T-cell responses than the subunit / oil-in-water OA composition.

ί.Ό8 T-cell responses - FIG. 17, bottom

RVMS from 24 mice per group were collected on the 7th day after immunization and tested in 1 pool / group. Inactivated trivalent whole viruses (1 μg / ml) were used as antigen for repeated stimulation.

As for the whole CE1 + P8 virus-specific CE8 + T cells expressing ΙΕ-2, ΙΕΝ-γ or both cytokines (Fig. 17, lower part):

the reference point in this experiment was relatively high due to the high background observed for PB8 of the negative control group;

however, higher specific T.sub.8 T cell responses were observed in mice immunized with trivalent split / A803 + MPB compared with other vaccine compositions.

LEARNING 3. A summary of the results and conclusions.

The following results were obtained:

1) P1i-specific CE4 + T cells detected as a result of TC8 on the 7th day after immunization showed the following:

(a) similar responses were obtained for P1iab compared to P1iap;

(b) the adjuvant composition induced a higher immune response compared to the non-adjuvant vaccine in the case of the influenza split vaccine (as observed in humans) and the subunit (Addra1) vaccine (not evaluated in humans). Adjuvant A803 in the form of an oil-in-water emulsion supplemented with MPB (groups 4 and 9) gave higher responses than adjuvant A803 in the form of an oil-in-water emulsion (groups 3 and 8);

(c) there is a tendency to higher CE4 responses with split / A803 + MPB compared to split / A803 (Fig. 17);

(d) the responses induced by the split vaccine were superior to those obtained with the subunit vaccine (cf. groups 1–4 and groups 5–9);

(e) a split vaccine supplemented with A803 adjuvant with or without MPB (groups 3 and 4) showed higher ί.Ό4 + T-cell responses than the subunit vaccine, both P1iab (group 5) and Addpra1 + OA ( group 7);

2) P1i-specific CE8 + T cells detected as a result of [Cg on the 7th day after immunization did not show any observed differences between split / A803 and simple split vaccine (as observed in humans). There was a tendency for a higher ί.Ό8 + T-cell response when using split / A803 + MPI compared to split / A803 or a simple split vaccine;

3) for the same adjuvant and for three strains, similar ΗΙ-titers were induced under the action of a subunit vaccine and a split vaccine. For the three strains, higher statistically significant titers were observed when the P1i vaccine (subunit or split) was supplemented with the A803 or A803 + MPB adjuvant compared to the simple P1i vaccine (the P1i vaccine OA is better than the simple P1i vaccine only for strain A / Auotshd).

An example of USH. A clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus and A803 with or without MPI as adjuvant in an elderly population over 65 years of age.

CS.1. Study plan.

Phase Ι An open randomized controlled trial in the elderly

- 55 011419 adults over 65 years of age (at least 65 years old) to evaluate the reactogenicity and immunogenicity of influenza vaccine candidates from C1axo8tyBK11pe Vyuodyuak containing adjuvant A803 or A803 + MPB administered intramuscularly, compared with the E1iapkh ™ vaccine (known in Belgium KTH ™).

Three parallel groups were considered:

one group of 50 subjects receiving 1 dose of reconstituted and A803-adjuvanted 8U influenza vaccine (E1AA803);

one group of 50 subjects receiving 1 dose of reconstituted and A803 + MPB adjuvant 8U influenza vaccine (E1AA803 + MPB);

one control group of 50 subjects receiving 1 dose of E1iaph ™ (E1iaph).

US.2. Vaccine composition and administration.

The strains used in the three vaccines were strains that were recommended by WHO for the northern hemisphere for the 2004-2005 season, i.e. Λ / No \ ν Ca1ebosha / 20/99 (ΉΙΝΙ), Λ / No \ ν Ca1jotta / 3/2003 (NoN2) and B / Lapdki / 10/2003. Like E1iaph ™ / a-K1x ™, a commercially available vaccine used for comparison, adjuvant vaccines (with A803 or A803 + MPB) contain 15 μg of hemagglutinin (HA) per strain of influenza virus per dose.

Adjuvant influenza vaccine candidates are 2-component vaccines consisting of concentrated trivalent inactivated cleaved virion antigens presented in a type стеклян glass vial and a type Ι pre-filled glass syringe containing adjuvant (A803 or A803 + MPB). They are made as described in detail in Example ΙΙ. The three inactivated cleaved virion antigens (monovalent bulk form) used in the adjuvanted influenza vaccine formulations are exactly the same as the active ingredients used in the preparation of the commercially available B1iapx ™ / a-K1x.

A803 Adjuvant Vaccine

The A803 adjuvant influenza vaccine candidate is a 2-component vaccine consisting of concentrated trivalent inactivated cleaved virion antigens presented in an Ι type glass vial (335 μl) (antigen container) and a Ι type prefilled glass syringe containing 8B62 emulsion (335 μl) (container for adjuvants). The description and preparation of the A803-containing candidate vaccine is explained in Example ΙΙΙ. A803 + MP-Adjuvant Vaccine

Briefly, the A803 + MPP adjuvanted influenza vaccine candidate is a 2-component vaccine consisting of concentrated trivalent inactivated cleaved virion antigens presented in a Ι type glass vial (335 μl) (antigen container) and a Ι type prefilled glass syringe containing adjuvant A803 + MPB (360 μl) (container for adjuvants). At the time of injection, the contents of the antigen container are removed from the vial using a syringe containing the A803 + MPB adjuvant, followed by gentle mixing of the contents of the syringe. Before injection, the used needle is replaced with an intramuscular injection needle and the volume is adjusted to 530 μl. 1 dose of reconstituted A803 + MPB-adjuvanted influenza vaccine candidate corresponds to 530 μl. To obtain 15 μg of HA for each strain of the influenza virus during the restoration of the A803 + MPB adjuvant vaccine, inactivated cleaved virion antigens are 2 times more concentrated in the antigen container (i.e. 60 μg of HA / ml) compared to B1iapch ™ (t e. 30 μg HA / ml).

The composition of a 1 dose reconstituted adjuvanted influenza vaccine is identical to that given in table. 45 (see Example X1), with the exception of influenza virus strains. Both vaccines were administered intramuscularly.

CS 3. The challenge, endpoints and results regarding SM!

The tasks with respect to CMI were to determine which of the compositions: the immunogenic composition supplemented with the A803 or A803 + MPB adjuvant, and the immunogenic composition without any adjuvant, has the highest immunostimulating activity on 0.Ό4- and 0Ό8-mediated immunity in individuals vaccinated antigens of influenza virus.

CS 3.1. Endpoints and results regarding SM!

Observed variable

On the 0 and 21 days: the frequency of occurrence of cytokine-positive CO4 / SE8 cells by 10 ⁶ for five different cytokines. In each test, the response of 0Ό4 / 0Ό8 T cells to the pool of the following three antigens was quantitatively calculated: antigen No. \ ν Ca1ebosha, antigen Vuot ^ ηд, Lapdka antigen.

Secondary variables

Antigen-specific 0Ό4 and 0Ό8 T-cell response, expressed in five different tests:

1) cells producing at least two different cytokines (0'040 ^ W-2, ΙΕΝγ, ΤΝΕα);

- 56 011419

2) cells producing at least CO40b and another cytokine (ΙΕ-2, ΤΝΕα, ΙΕΝγ);

3) cells producing at least ΙΕ-2 and another cytokine (СО40Ь, ΤΝΕα, ΙΕΝγ);

4) cells producing at least ΙΕΝγ and another cytokine (ΙΕ-2, ΤΝΕα, СО40Ь);

5) cells producing at least ΤΝΕα and another cytokine (ΙΕ-2, СО40Ь, ΙΕΝγ).

CΜI response analysis

A CI analysis was performed for the entire vaccinated cohort.

(a) For each group treated, the frequency of occurrence of T-lymphocytes secreting in response to ί.Ό4 / ί.Ό8 was determined for each vaccinated group, for each time point (day 0, day 21) and for each antigen: Νο \ ν Ca1ebosha, Vuot ^ ηd and Lapdsh and a pool of three different strains.

(b) Descriptive statistics of the individual difference between responses to time points (GoMΡιό) for each vaccinated group and each antigen for each of five different cytokines.

(c) Comparison of three groups in relation to five different cytokines in

CE4 T-cell response to No. \ ν Ca1ebosh, ’No. patches, Lapdki and pool of three strains,

SE8 T-cell response to No. \ ν Ca1ebosha, ’No. patches, Lapdki and pool of three strains.

(d) To compare the differences found between the three groups, a non-parametric test (Kruskal-Wallis test) was used and the statistical value p was calculated for each antigen for each of five different cytokines.

(e) The Wilcoxon test was used to test the pairwise comparison between the two groups, respectively, Ε1i-Α803 + ΜΡ ^ versus Ε1ia ^^ x, Ε1и-Α803 + ΜΡ ^ versus Ε1ι.ι-Α803 and Ε1ι.ι-Α803 versus Ε1ia ^^ x.

(e) All criteria of significance were bilateral. P values of less than or equal to 0.05 were considered statistically significant.

CS.3.2. CME Results

The results were expressed as the frequency of occurrence of cytokine (s) -positive SE4 or SE8 T cells within the SE4 or SE8 T cell subpopulation.

The frequency of occurrence of antigen-specific SE4 T-lymphocytes (a) The frequency of occurrence of antigen-specific CE4 T-lymphocytes secreting in response was determined for each vaccinated group, at each time point (day 0, day 21) and for each antigen ( pool, No. \ ν Sa1ebosha, \ ν \ Όΐηίπ§ and Lapdki) by analogy with how was carried out in example ΙΙΙ.

(b) When comparing the differences in the frequency of occurrence of antigen-specific CE4 T lymphocytes between the three groups according to the Kruskal-Wallis test, it is seen that all p values were less than 0.05 and were considered statistically significant.

(c) When comparing the differences in the frequency of occurrence of antigen-specific CE4 T-lymphocytes between the Ε1i-Α803 + ΜΡ ^ and Ε1ia ^^ x groups according to the Wilcoxon test, it is seen that all p values were less than 0.05 and were considered statistically significant.

(d) When comparing the differences in the frequency of occurrence of antigen-specific CE4 T-lymphocytes between the Ε1ι.ι-Α803 and Ε1ia ^^ x groups according to the Wilcoxon test, it is seen that all p values were less than 0.05 and were considered statistically significant.

(e) When comparing the differences in the frequency of occurrence of antigen-specific CE4 T-lymphocytes between the Ε1ι.ι-Α803 and Ε1и-Α803 + ΜΡ ^ groups according to the Wilcoxon test, it is seen that all p values exceeded 0.05 and were not considered statistically significant.

Individual difference between time points (Roy-rge) in CE4 T-lymphocytes (a) Descriptive statistics of individual differences between time points (ΡΟ8Τ-ΡΚΕ) in CE4 T-lymphocyte responses were calculated for each vaccinated group and for each antigen for each of five different cytokines by analogy with how made in example ΙΙΙ.

(b) When comparing the individual differences of ΡΟ8Τ-ΡΚΕ in the responses of antigen-specific CE4 of T-lymphocytes between the three groups according to the Kruskal-Wallis test, it is seen that all p values were less than 0.001 and they were considered highly statistically significant.

(c) When comparing the individual difference ΡΟ8Τ-ΡΚΕ in the responses of antigen-specific CE4 of T-lymphocytes between Ε1i-Α803 + ΜΡ ^ and Ε1ia ^^ x using the Kruskal-Wallis test, it is seen that all p values were less than 0.05 and they were statistically calculated meaningful.

(d) When comparing the individual difference ΡΟ8Τ-ΡΚΕ in the responses of antigen-specific CE4 of T-lymphocytes between Ε1π-Α803 and Ε1ia ^^ x using the Kruskal-Wallis test, it is seen that all p values were less than 0.001 and were considered highly statistically significant.

(e) When comparing the individual difference ΡΟ8Τ-ΡΚΕ in the responses of antigen-specific CE4 of T-lymphocytes between Ε1i-Α803 + ΜΡ ^ and Ε1π-Α803 using the Kruskal-Wallis test, it is seen that all p values were more than 0.05 and were not considered as statistically significant.

CS 4. Objective, endpoints, and results with respect to the Β-cell secondary immune response.

The objective of the study was to study the possibility of inducing a significant frequency of occurrence of memory B cells specific for the Ε1 and antigen after a single intramuscular vaccination with the υ1υ candidate vaccine containing the Α803 + ΜΡ ^ or Α803 adjuvant, compared with Ε1ia ^^ x in elderly people. The frequency of occurrence of memory B-cells was evaluated according to the Ε1 ^ 8poΐ analysis of B-cells.

- 57 011419 νΙΙΙ. 4.1. Endpoints for a B-cell secondary immune response.

Endpoints:

(a) on day 0, day 21: cells producing cultured ΐη νίΐτο B-cells of memory, measured according to the I L-analysis of B-cells, in all subjects in terms of the frequency of occurrence of a specific antigen-specific plasma per million (10 ⁶ ) ΙβΟ-producing plasma cells;

(b) the difference between the states after (21st day) and before (0th day) vaccinations was also expressed as the frequency of occurrence of cells that form antibodies specific for the influenza virus per million (10 ⁶ ) antibody-forming cells.

νΙΙΙ.4.2. Results for a B-cell secondary immune response.

The frequency of occurrence of influenza virus-specific antibody-forming cells per million (10 ⁶ ) antibody-forming cells was determined. The results showed that the frequency of occurrence of memory B cells specific for the II antigen between the groups A1i-A303 + MPL and A1iag1x, according to the Wilcoxon criterion, was significantly (p less than 0.05) higher for strain B / Lap8, but not for two other strains (strains Α \ at Sakata and Vuot ^ ηβ).

The individual difference between the time points (ροδ р-рге) was also determined for memory B cells specific for the H1i antigen. The results showed that the individual difference between the time points (ροδΐ-рге) in the frequency of occurrence of memory B cells specific for the H1i antigen between the Hai-A803 + MPB and Haiapch groups according to the Kraskel-Wallis test was significant (p less than 0.05 ) higher for strain B / Lapji, but not for the other two strains (strains Α \ e SaksYata and Vuot ^ ηβ).

The results are shown in FIG. eighteen.

Example ΙΧ. Preclinical evaluation of adjuvant and non-adjuvant ferret influenza vaccines (study ΙΙΙ).

ΙΧ.1. Justification and objectives.

In this study, the commercially available influenza trivalent split vaccine OZK, either the non-adjuvant (Yaialx ™) or the adjuvant A803 + MRL, was compared with two other commercially available subunit vaccines:

Я1иаб ™, adjuvant subunit vaccine from Chygop (adjuvant is adjuvant MJ59 from Chygop),

Agpr1 ™, a commercially available non-adjuvant subunit vaccine for (ΊιίΐΌΐι, which was supplemented in this study with the adjuvant AZ03.

The objective of this experiment was to assess the ability of these vaccines to reduce disease symptoms (body temperature) and viral shedding in nasal secrets of ferrets subjected to control infection with heterologous strains.

The following endpoints were determined:

1) primary endpoint: reduction of viral shedding in nasal washes after heterologous control infection;

2) minor endpoints: analysis of the humoral response by means of AL and monitoring of temperature near the priming point and heterologous control infection.

ΙΧ. 2. Experiment plan.

ΙΧ.2.1. Processing / group.

Female ferrets (Miig! E1a riGOpiz 1igo) at the age of 14-20 weeks were received from MPZAU ^ n ^ Napsu (11thrz1ngene ', IR). Ferrets were primed intranasally on the 0th day with the heterosubtypic strain Η1Ν1 A / ZUSk ^ t ^ / ED (4 1ο§ TSGO ₅₀ / ml). On the 21st day, a full human dose (1 ml of the vaccine dose, 15 μg HA / strain) of the combination Η1Ν1 A / \ e \ x Ca1еάοη ^ а / 20/99, Η3Ν2 Α / Вуοт ^ ηβ / 3 / was administered to the ferrets by intramuscular injection 2003 and B / Lapji / 10/2003. Then the control ferrets were infected on the 42nd day via the intranasal route with the heterotypic strain Η3Ν2 A / Rapata / 2007/99 (4.51 1ts TSGO ₅₀ / ml). Groups (6 ferrets / group) are given in table. 41. The reading results are detailed in table. 42.

Table 41

Corpse- Antigen (s) + pa dosage

Trivalent simple (Yaiapkh ™) Trivalent AZOZ + MRI.

E1ia0 ™

Adprra! ™ AZOZ

Composition + Dosage

Full Ηϋ: 15 mcg HA / strain

Full Ηϋ:

mcg HA / strain Full Ηϋ:

mcg HA / strain Full Ηϋ:

μg HA / strain

Notes (mode / path / infection) in / m; 21st day in / m; 21st day in / m; 21st day in / m; 21st day

Other treatments

Priming Η1Ν1 (A / 81oscpo1t / 24/90) on day 0 _________

Priming Η1Ν1 (A / 81oskobo1t / 24/90) on day 0 ________

Priming Η1Ν1 (A / 81oscpo1t / 24/90) on day 0 ________

Priming Η1Ν1 (A / 81oscpo1t / 24/90) on day 0

- 58 011419

ΙΧ.2.2. Preparation of vaccine compositions.

Trivalent simple (non-adjuvant) split composition: preparation for 1 ml Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and \ 'E8 (in quantities including detergents present in the strains) are added to water for injection. The amount of detergents reaches the following values: 375 μg Tween 80, 55 μg Triton X-100 and 50 μg \ 'E8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and 17.5 μg of strain B are added, each time with 10 minutes stirring between additions. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

Trivalent adjuvant A803 + MRP split composition: preparation for 1 ml Tenfold concentrated PB8 (pH 7.4 based on a single concentration), as well as a mixture containing Tween 80, Triton X-100 and \ 'E8 (in quantities, taking into account detergents present in the strains) are added to water for injection. The amount of detergents reaches the following values: 375 μg Tween 80, 55 μg Triton X-100 and 50 μg \ 'E8 per 1 ml. After 5 minutes of stirring, 15 μg of each strain Η1Ν1, Η3Ν2 and B are added each time with 10 minutes stirring between additions. After 15 minutes of stirring, 250 μl of 8B62 emulsion (prepared as described in detail in Example No. 1.) is added. The mixture was again stirred for 15 minutes immediately before the addition of 25 μg MPB. The composition is stirred for 15 minutes at room temperature and stored at 4 ° C, unless administered immediately.

NiAi ™ composition: preparation for 1 ml

Two-fold dilution of NiAi ™ vaccine is carried out in a PB8 pH 7.4 buffer.

Composition Adprra1 ™ A803: preparation for 1 ml

250 μl of pH 7.4 PB8 buffer was added to 1 dose of Addpra1 ™. After stirring, add 250 μl of 8B62 emulsion (prepared as described in detail in Example A.1.). The mixture was stirred at room temperature.

ΙΧ.2.2. Reading results.

Table 42

Reading result Time point Sample Type 1p / Po Analysis Method Viral shedding 3 days before and within 7 days after priming For 1 day up to 5 days after infection control Nasal swabs 1p Titration l _ / »l  L | -D · ____ _ ινιυπιπ ι irin1 t ° aa about yi ι ik di and in ι sccmis h days after priming 2 days before and within 4 days after infection control IM11 Lsi-11 α ι in the peritoneal cavity III ι elsme | ri> 1 ΙΗΑ Before, after priming, after immunization, after challenge Serum 1p ΙΗΑ

Ιιι = individually / Po = pool.

ΙΧ. 3. Results (Figs. 19-22).

ΙΧ.3.1. Temperature monitoring.

Individual temperatures were recorded using sensors and telemetry recordings. All implants were checked and updated and a new calibration was performed using Ό8Ι before placement in the intraperitoneal cavity. At the time of these measurements, all animals were individually housed in separate cages. The temperature was recorded every 15 min, starting from 2 days before the control infection and up to 4 days after the control infection, and the average value was calculated in the middle of the day. The results are shown in FIG. nineteen.

results

After challenge, a peak in body temperature was observed after immunization of the ferrets with a non-adjuvant (simple) trivalent split- (G1iaph ™) or Nyai ™ subunit vaccine (which contains an MT59 oil-in-water emulsion). No peak was observed either after immunization of ferrets with a trivalent split vaccine with A803 + MPB adjuvant, or after immunization of ferrets with subunit Adpr1 ™ with A803 adjuvant. Conclusion: The additional benefits of A803-containing vaccines in preventing body temperature rise after challenge for both tested split and subunit vaccines have been demonstrated, as opposed to the inability of MT59-containing vaccines to prevent this temperature rise in ferrets after challenge.

ΙΧ.3.2. Viral shedding.

Titration of viruses in nasal washes was performed on 6 animals per group. Nasal meanings

- 59 011419 you received by introducing 5 ml of PB8 into both nostrils of awake animals. The inoculum was collected in a Petri dish and placed in sample containers on dry ice (-80 ° C).

All nasal samples were first filtered under sterile conditions through 8px X filters (Soiag) to remove any bacterial contamination. 50 μl of serial ten-fold dilutions of nasal washings were transferred to microtiter plates containing 50 μl of medium (10 wells / dilution). Then, 100 μl of MOSCOW cells (2.4 × ^{10 5} cells / ml) were added to each well and incubated at 35 ° C for 5-7 days. After 5-7 days of incubation, the culture medium is carefully removed, 100 μl of 1/20 ^ 8T-1-containing medium are added and incubated for another 18 hours.

The intensity of the yellow formazan dye resulting from the restoration of ^ 8T-1 by viable cells is proportional to the number of viable cells present in the well by the end of the virus titration analysis and is quantified by measuring the absorbance at the corresponding wavelength (450 nm). The reference point is defined as the average OO value for uninfected control cells — 0.3 OO (0.3 OO correspond to +/- 3 80 (standard deviation) OO for uninfected control cells). A positive score is determined when the TOE is below the reference point, and, conversely, a negative score is determined when the TOE is above the reference point. Viral shedding titers were determined according to Kei apy MiepsN and expressed as 1ode TCU ₅₀ / ml.

results

The results are shown in FIG. 20. Weaker viral shedding was observed after challenge with a trivalent split vaccine with A803 + MPB adjuvant or AdprA1 ™ subunit vaccine with A803 adjuvant compared to the very slight decrease in viral shedding observed after immunization of ferrets with a non-adjuvant (simple) three and one ™) or P1iay ™ subunit vaccine.

By analogy with what was discussed with respect to raising body temperature, the additional benefit of A803-containing vaccines was observed compared to MP59-containing vaccines.

ΙΧ.3.3. ΗΙ-Captions.

Anti-hemagglutinin antibody titers of influenza virus Η3па2 were determined using a hemagglutination inhibition test (ΗΙ). The principle of the теста-test is based on the ability of specific anti-influenza antibodies to inhibit the hemogglutination of red blood cells (CV C) of chicken with hemagglutinin (HA) of the influenza virus. Serum samples were initially treated with a 25% solution of neuraminidase (CFU) and heat inactivated to remove non-specific inhibitors. After pretreatment, two-fold dilutions of serum were incubated with 4 units. hemagglutination of each strain of influenza virus. Then chicken red blood cells were added and inhibition of agglutination was evaluated. The titers were expressed as the reciprocal of the highest dilution of serum at which complete inhibition of hemagglutination was observed. Since the first dilution of serum was 1:10, an undetectable level was evaluated as a titer of 5.

results

After immunization with Η3Ν2 A / ^ wattd, higher humoral responses (тит-titers) were observed in ferrets immunized with the trivalent split vaccine with A803 + MPB adjuvant or AdprA1 ™ subunit vaccine with A803 adjuvant, compared with the humoral adjuvant (simple) trivalent split vaccine (P1iaph ™) or subunit vaccine P1iay ™ (Fig. 21).

After immunization with Η3Ν2 A / ^ wattd, higher humoral responses (тит-titers) were also observed against the drifting strain Η3Ν2 A / Rapata, used as a strain for experimental infection, in ferrets immunized with the trivalent split vaccine with A803 + MP1 adjuvant or Ad1 adjuvant with adjuvant A803, compared with the humoral response of ferrets immunized with a trivalent simple split vaccine or P1iay (Fig. 22).

This cross-reaction observed with the adjuvant proposed by the authors (A803 or A803 + MPB) against the heterologous strain correlated with the protection observed in ferrets immunized with the trivalent split vaccine with the A803 + MPB adjuvant or the AdprA1 ™ subunit vaccine with the adjuvant and the adjuvant infection with this heterologous strain. This cross-reactivity to the heterologous strain induced by A803-containing vaccines was not induced by MP59-adjuvanted vaccines (P1iAi ™).

Example X. Clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus and adjuvant A803 with or without WIDES, in an elderly population over the age of 65: data on the persistence of immunogenicity on the 90th and 180th days.

X.1. Study plan.

Phase Ι of an open randomized controlled trial in the elderly over 65 years of age (at least 65 years old) to assess the reactogenicity and immunogenicity of anti-influenza candidate vaccines from S1axo8tTs11K1te Vu1od1sak containing adjuvant A803 or A803 + MP1 administered intramuscularly, compared with ™ (known in Belgium as α

- 60 011419

Κιχ ™). This study corresponds to what is set forth in the νΙΙΙ example.

Three parallel groups were considered:

one group of 50 subjects receiving 1 dose of reconstituted and A803-adjuvanted 8ν influenza vaccine (P1 and A803);

one group of 50 subjects receiving 1 dose of reconstituted and A803 + MPB adjuvanted 8ν influenza vaccine (P1AA803 + MPB);

one control group of 50 subjects receiving 1 dose of P1iaph ™ (P1iaph).

X.2. Immunogenicity results.

X.2.1. Endpoints and results for a humoral immune response.

In order to evaluate the humoral immune response induced by A803 and A803 + MPB adjuvant vaccines, and its persistence, the following parameters were calculated for each treatment group.

On days 0, 21, 90 and 180: serum antibody hemagglutination inhibitory (ΗΙ) titers tested individually against each of the three influenza virus strains presented in the vaccine (anti-CMM, anti-113 2 and anti-B antibodies).

OMT of serum антител antibodies with 95% CI on days 0, 21, 90 and 180.

Seroconversion levels from 95% CI on the 21st, 90th and 180th days.

Conversion factors from 95% CI on day 21.

Seroprotection levels from 95% CI on days 0, 21, 90 and 180.

results

OMT for ΗΙ antibodies with 95% CI are shown in FIG. 23. Before vaccination, OMT antibodies for all three vaccine strains were in the same range for the three groups. After vaccination, anti-hemagglutinin antibody levels increased significantly. However, after vaccination, the OMT antibodies for the three vaccine strains remained within the same ranges for all vaccines. On the 21st day, a weak tendency in favor of two adjuvant vaccines compared to P1iapx was observed for strains of AJ ^ Ca1syosha and B / Lapji, and among these two adjuvant vaccines, higher OMT were observed with Pb-A803 for strains Azhuott and B / Lapzi.

The same trends were observed on the 90th day. On the 180th day, OMT antibodies for three vaccine strains were within the same ranges for three vaccines.

All influenza vaccines met the requirements of European authorities for the annual registration of inactivated influenza vaccines | Ho1c £ og ш й ап аг тот аг тот тот оп о о о о с р р р р р р [[[[п П исп исп / ст ст ст ст 1!!!!! o £ appia1 §1tat syapdsz (CPMP / B ^ P / 214/96)] for subjects over the age of 60.

3 months (90 days) and 6 months (180 days) after vaccination, seroprotection levels were still above the minimum level of 60% required by European authorities for any study group under consideration. On the 90th day, the minimum level of seroconversion of 30%, required by European authorities, was still achievable for all vaccine strains in the three vaccine groups, with the exception of P1iapch for the strain АЖ ^ Ca1syosh. On the 180th day, it was still achievable using three vaccines for the strains Azhuottd and V / Lapdzi, but not for the strain AJ ^ Ca1syosha (Tables 43 and 44).

Table 43

Seroprotection levels as a percentage of those vaccinated with serum titers of hemagglutination inhibition greater than or equal to 1:40 (ATP (according to the protocol) -cohort for immunogenicity)

> 1:40 95% CI Antibody Group Schedule Ν η % IX be Α / Νβνν Ca1edop1a E1iA803 + MP1_ REE fifty 28 56.0 41.3 70.0 ΡΙ (ϋ21) fifty 46 92.0 80.8 97.8 ΡΙ (ϋ90) fifty 43 86.0 73.3 94.2 ΡΙ (ϋ180) fifty 39 78.0 64.0 88.5 Yayaph REE fifty 26 52.0 37,4 66.3 ΡΙ (ϋ21) fifty 46 92.0 80.8 97.8 ΡΙ (ϋ90) fifty 38 76.0 61.8 86.9 ΡΙ (ϋ180) fifty 34 68.0 53.3 80.5 E1iA803 REE 49 28 57.1 42,2 71.2 ΡΙ (ϋ21) 49 48 98.0 89.1 99.9 ΡΙ (ϋ90) 49 45 91.8 80,4 97.7 ΡΙ (ϋ180) 49 38 77.6 63,4 88.2 ALL / wat1pd E1iA803 + MP1_ ΡΕΕ fifty 33 66.0 51,2 78.8 ΡΙ (ϋ21) fifty 47 94.0 83.5 98.7 ΡΙ (ϋ90) fifty 46 92.0 80.8 97.8 ΡΙ (ϋ180) fifty 45 90.0 78,2 96.7 E1iaph ΡΕΕ fifty 32 64.0 49.2 77.1 ΡΙ (ϋ21) fifty fifty 100 | 92.9 100.0

- 61 011419

ΡΙ (ϋ90) fifty 49 98.0 89.4 99.9 ΡΙ (ϋ180) fifty fifty one hundred 92.9 100.0 NIAZOZ PPE 49 34 69,4 54.6 81.7 ΡΙ (ϋ21) 49 48 98.0 89.1 99.9 ΡΙ (ϋ90) 49 46 93.9 83.1 98.7 ΡΙ (ϋ180) 49 47 95.9 86.0 99.5 W / Lapji NiA303 + MP1_ RKE fifty nineteen 38,0 24.7 52.8 ΡΙ (ϋ21) fifty fifty one hundred 92.9 100.0 ΡΙ (ϋ90) fifty 47 94.0 83.5 98.7 ΡΙ (ϋ180) fifty 46 92.0 80.8 97.8 Niaph ΡΚΕ fifty 17 34.0 21,2 48.8 ΡΙ (ϋ21) fifty 48 96.0 86.3 99.5 ΡΙ (ϋ90) fifty 47 94.0 83.5 98.7 ΡΙ (ϋ180) fifty 47 94.0 83.5 98.7 NIAZOZ ΡΚΕ 49 25 51.0 36.3 65.6 ΡΙ (ϋ21) 49 49 one hundred 92.7 100.0 ΡΙ (ϋΘΟ) 49 47 95.9 86.0 99.5 ΡΙ (ϋ180) 49 46 93.9 83.1 98.7

Ν = number of subjects with available results.

p /% = number / percentage of subjects with a titer in a specific range.

PKE = titer before vaccination.

ΡI (^ 21) = blood sampling after vaccination on day 21.

ΡI (^ 90) = blood sampling after vaccination on day 90. ΡI (^ 180) = blood sampling after vaccination on day 180.

Table 44

The seroconversion level for hemagglutination-inhibiting antibody titers (ΗΙ), defined as the percentage of vaccinees who have at least a four-fold increase in serum тит titer, for each time point after vaccination compared to 0 days (ATP cohort for immunogenicity)

Vaccine strain Schedule Group N 4x η % 95% CI ίί be Α / ΝΕνν ΟΑΙ_ΕϋΟΝΙΑ 21st day NiA303 + MP1 fifty thirty 60.0 45,2 73.6 Niaph fifty 25 50,0 35.5 64.5 NIAZOZ 49 31 63.3 48.3 76.6 90th day NiA303 + MP1 fifty nineteen 38,0 24.7 52.8 Niaph fifty 14 28.0 16,2 42.5 NIAZOZ 49 17 34.7 21.7 49.6 180th day NiA303 + MP1 fifty 12 24.0 13.1 38,2 Niaph fifty eleven 22.0 11.5 36.0 NIAZOZ 49 10 20,4 10,2 34.3 ΑΜ / ΥΟΜΙΝΘ 21st day NiA303 + MP1 fifty 46 92.0 80.8 97.8 Niaph fifty 38 76.0 61.8 86.9 NIAZOZ 49 40 81.6 68.0 91.2 90th day NIAZOSIS + MRI fifty 33 66.0 51,2 78.8 Niaph fifty 33 66.0 51,2 78.8 NIAZOZ 49 31 63.3 48.3 76.6 180th day NIAZOSIS + MRI fifty 27 54.0 39.3 68,2 Niaph fifty 23 46.0 31.8 60.7 NIAZOZ 49 26 53.1 38.3 67.5 Β / ΰΐΑΝ08υ 21st day NIAZOSIS + MRI fifty 44 88.0 75.7 95.5 Niaph fifty 38 76.0 61.8 86.9 NIAZOZ 49 43 87.8 75,2 95.4 90th day NIAZOSIS + MRI fifty 37 74.0 59.7 85,4 Niaph fifty 36 72.0 57.5 83.8 NIAZOZ 49 37 75,5 61.1 86.7 180th day NIAZOSIS + MRI fifty 32 64.0 49.2 77.1 Niaph fifty 29th 58.0 43,2 71.8 NIAZOZ 49 31 63.3 48.3 76.6

Ν = number of subjects with available results both before and after vaccination. p /% = number / percentage of subjects with at least fourfold increase. 95% CI = strictly 95% confidence interval.

EE = lower limit, H = upper limit.

X.2.2. Endpoints and results for the CΜI response.

In order to evaluate the cellular immune response induced by adjuvant vaccines and its persistence, the following parameters were calculated for each treatment group.

- 62 011419

For each time point (days 0, 21, 90 and 180): the frequency of occurrence of cytokine-positive CO4 / CE8 cells by 10 ⁶ in different tests (antigens No. \ ν Ca1ebosha, HUuotnsch and Lapdts, accounted separately, as well as in the pool, on the 0th and 21st days; antigens Νο \ ν Sa1ebosha, ^ wattshd, Lapdkts and \ο \ ν Wagk, recorded separately, as well as in the pool, on the 90th and the 180th day).

All in two: cells producing at least two different cytokines (ί.Ό40Ε ΙΕΝ-γ, ΙΕ-2, 'ΓΝΕ-α). CO40: cells producing at least SE40B and another cytokine (ΙΕΝ-γ, ΙΕ-2, TXL-a). ΙΕΝ-γ: cells producing at least ΙΕΝ-γ and another cytokine (SP40b, ΙΕ-2, TXL-a). ΙΕ-2: cells producing at least ΙΕ-2 and another cytokine (ί.Ό40Ε ΙΕΝ-γ, TXL-a). ΪΝΕ-α: cells producing at least TNG-a and another cytokine (SP40b, ΙΕΝ-γ, ΙΕ-2). results

The following main results were obtained (Fig. 24):

(a) 21 days after vaccination, the frequency of occurrence of cytokine-positive CE4 T cells (ΙΕ-2, ί.Ό40Ε ΈΝΕ-α and ΙΕΝ-γ) was significantly higher in the two adjuvant vaccine groups compared to the Nnap.h. However, no significant difference between the two adjuvants was noted;

(b) all statistical differences between adjuvant vaccines and B1iap.x were maintained inclusively up to the 90th day and up to the 180th day with the following exceptions for the 180th day:

no statistically significant difference was found between B1iA803 / MRB and Nnap.kh for all two each, ί.Ό40Ε ΙΕΝ-γ and Ш2 (only HUuoshshd strain) and for all two, SO40B and TYT-a (only Νονν Wogk strain) ;

no statistically significant difference was found between B1nA803 and Nnap.x for GB2 (only Lapdcc strain);

(c) the absence of a statistically significant difference between the two adjuvant vaccines was confirmed on the 90th and 180th days inclusive;

(d) the difference between the states before and after vaccination (day 21) in the responses of CE4 T-lymphocytes for all cytokines studied (ΙΕ-2, ί.Ό40Ε ΕΝΕ-α and ΙΕΝ-γ) was significantly higher when using two adjuvant vaccines compared to Niap.x ™. However, no significant difference was observed between the two adjuvants;

(e) vaccination did not have any measurable effect on the SE8 response in any treatment group.

Example ΧΙ. A clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus and A803 adjuvant with MPI in an elderly population over the age of 65 years.

ΧΙ. 1. Research plan and objectives.

The Ι / ΙΙ phase of an open controlled study was carried out with the aim of assessing the reactogenicity and immunogenicity of the influenza candidate vaccine from S1axo8tLyKlpe Vu1odyu18, containing adjuvant A803 + MPB, in an elderly population over 65 years old (over 65 years old), previously vaccinated in 2004. the same candidate vaccine. For evaluations of immunogenicity and safety, the Niap.x ™ vaccine (known in Belgium as α-K ^ x ™) was used for comparison.

Two parallel groups were evaluated:

one group of approximately 50 subjects who had previously received 1 dose of reconstituted adjuvanted influenza vaccine in a previous clinical trial;

one control group (Nnap.kh) from approximately 50 subjects who had previously received 1 dose of Niap.x ™ in the previous clinical trial.

One of the objectives of this study was to evaluate the humoral immune response (anti-hemagglutinin and anti-MPI titers) to revaccination with the adjuvanted influenza vaccine Ε1υА803 + MPI, administered approximately 1 year after the first dose. For comparison purposes, subjects already treated with Niap.x ™ in a previous trial received a dose of a commercially available vaccine and formed a control group for this trial.

ΧΙ. 2. Vaccine composition and administration.

The strains used in the three vaccines were strains that were recommended by WHO for the northern hemisphere for the 2005-2006 season, i.e. A / Yed Sa1ebosha / 20/99 (Η1Ν1), A / Νονν Ca1Togsha / 7/2004 (Η3Ν2) and B / Lapdkts / 10/2003. Like Ε1iaπx ™ / α-K ^ x ™, a commercially available vaccine used for comparison, the A803 + MPD adjuvant vaccine (hereinafter for short, the adjuvant vaccine) contains 15 μg of hemagglutinin (HA) per strain of influenza virus per dose.

An adjuvant influenza vaccine candidate is a 2-component vaccine consisting of concentrated trivalent inactivated cleaved virion antigens presented in a type стеклян glass vial and a type енного prefilled glass syringe containing adjuvant A803 + MPL. They are made according to the method described in detail in example ΙΙ.

At the time of injection, the contents of a pre-filled syringe containing adjuvant are injected into a vial that contains concentrated trivalent inactivated cleaved virion antigens. After mixing, the contents are returned back to the syringe, and the needle is replaced with a needle for intramuscular injection. 1 dose of reconstituted adjuvanted influenza vaccine candidate corresponds to 0.7 ml. Candidate Adjuvant Influenza Vaccine Pre

- 63 011419 is a vaccine that does not contain a preservative.

The composition of a 1 dose reconstituted adjuvanted influenza vaccine is given in table. 45. Both vaccines were administered intramuscularly.

Table 45

Composition of the reconstituted influenza adjuvant (A803 + MPB) candidate vaccine

Component Quantity per dose Inactivated cleaved virions: - Α / Νθνν Ca1ec1ogpa / 20/99 (H1 N1); 15 mcg - Α / Νβνν Ca1 | (ogp1a / 7/2004 (Η3Ν2); 15 mcg - VALapji / 10/2003 15 mcg Adjuvant: Emulsion 8B62: - squalene, 10.68 mg - O1_-alpha-tocopherol, 11.86 mg - Polysorbate 80 (Twin 80); 4.85 mg MRI 25 mcg

Xt3. Immunogenicity results.

XT3.1. Endpoints and results for an anti-HA humoral immune response.

Observable variables

On day 0 and day 21: serum antibody hemagglutination (III) inhibitory titers tested individually against each of the three influenza virus strains presented in the vaccine (antiINN1, anti-H3J and anti-B antibodies).

Secondary variables (with 95% confidence intervals) geometric mean titers (OMT) of serum W-antibodies with 95% confidence intervals (95% CI) before and after vaccination, seroconversion levels * from 95% CI on day 21, seroconversion factors * * with 95% CI on day 21, seroprotection levels *** with 95% CI on day 21.

* Seroconversion rate, defined as the percentage of vaccinees, either with a W-titer before vaccination <1:10 and a titer after vaccination> 1:40, or with a titer before vaccination> 1:10 and at least a four-fold increase in titer after vaccination, for each vaccine strain.

** Seroconversion factor, defined as a multiple increase in serum OMT III on day 21 compared with day 0 for each vaccine strain.

*** The level of protection, defined as the percentage of vaccinees with a serum W-titer> 40 after vaccination (for each vaccine strain), which is usually taken as an indicator of protection.

results

As expected, before vaccination, the vast majority of subjects in both groups were already seropositive for the three strains. In two groups, OMT before vaccination for all three vaccine strains were in the same range. There was a tendency to higher OMT after vaccination for all three vaccine strains in the E1i-A803 + MPB group compared to the Niapch group, although 95% CI overlap was observed (Fig. 25).

Two influenza vaccines met the requirements of the European authorities regarding the annual registration of inactivated influenza vaccines 60 years (tab. 46).

Table 46

Seroprotection levels, seroconversion levels and conversion factors on the 21st day (ATP cohort by immunogenicity)

Strains Group N Seroprotection Level (ΗΙ-titer> 40)% Seroconversion Level (> 4x increase) [95% CI]% Seroconversion Factor [95% CI]% EU Standard (> 60 years old) > 60% > 30% > 2.0 Α / Νβνν Ca1ebop1'a Yai + MRYAZOZ 38 89.5 [75.20-97.06] 31.6 [17.5-48.7] 3.1 [2.2-4.4] Yayaph 45 82.2 [67.95-92.00] 31.1 [18.2-46.6] 2.5 [1.8-3.5] Α / Νβνν Wogk (Η3Ν2) Yai + MRI-AZOZ 38 92.1 [78.62-98.34] 78.9 [62.7-90.4] 8.8 [6.1-12.5] Yayaph 45 95.6 [84.85-99.46] 68.9 [53.4-81.8] 6.0 [4.4-8.3] W / Lapji (IN) Yai + MP1-A303 38 100 [90.75-100] 57.9 [40.8-73.7] 5.1 [3.7-7.0] Yayaph 45 100 [92.13-100] 37.8 [23.8-53.5] 3.1 [2.4-4.0]

- 64 011419

Ν = total number of subjects.

% = percentage of subjects with a titer on day 21 within a specific range.

CI = confidence interval.

Example ΧΙΙ. A clinical trial of a vaccine containing an antigenic preparation of cleaved influenza virus with adjuvant A803 and ΜΡΕ in two different concentrations in an elderly population over the age of 65 years.

ΧΙΙ. 1. Research plan and objectives.

The Ι / ΙΙ phase of an open randomized study to demonstrate the equally effective cell-mediated immune response of S1axo8thyK1te Vyuhschak influenza vaccines containing various adjuvants administered to the elderly (65 years of age and older), compared with P1iaph ™ (known in Belgium as a-CTH ™), administered to adults (18-40 years old).

Four parallel groups were evaluated:

(a) 75 adults (aged 18–40 years) in one control group receiving 1 dose of P1iapkh ™ (P1iapkh group);

(b) 200 elderly subjects (aged 65 years and older) randomized to three groups in a ratio of 3: 3: 2:

one group of 75 subjects receiving an influenza vaccine with A803 + MGE adjuvant (concentration 1-25 μg), one group of 75 subjects receiving an influenza vaccine with A803 + MGE adjuvant (concentration 2-50 μg),

P1i comparison group from 50 subjects receiving 1 dose of P1iapkh ™.

Paramount task

The paramount task is to demonstrate no less efficacy 21 days after vaccination with influenza adjuvant vaccines given to elderly subjects (aged 65 years and older), compared with P1iapkh ™ administered to adults (aged 18-40 years), with respect to the frequency of occurrence of influenza-specific ί.ί4 T-lymphocytes producing at least two different cytokines (ί.Ό40Ε Gb-2, ΕΝΡ-α, ΙΡΝ-γ).

Minor Tasks

Minor Tasks:

(a) to evaluate the safety and reactogenicity of vaccination with influenza adjuvant candidate vaccines within 21 days after intramuscular administration of the vaccine to elderly subjects (aged 65 years and older). For comparison, use P1iaph ™;

(b) evaluate the humoral immune response (anti-hemagglutinin titer) after 21, 90, and 180 days after vaccination with influenza adjuvant candidate vaccines. For comparison, use P1iaph ™.

Third task

The third task is to assess the cell-mediated immune response (production of ΙΡΝ-γ, ΙΕ-2, СО40B and ΊΝΕ-α and the response of memory B cells) 21, 90 and 180 days after vaccination with adjuvanted influenza vaccines. For comparison, use P1iaph ™.

ΧΙΙ. 2. Vaccine composition and administration.

The influenza vaccine system with adjuvant A803 + MGE (25 μg per dose) is also used in the study illustrated in Example ΧΙ. The influenza vaccine system with A803 + MGE adjuvant (50 μg per dose) is an identical composition, except that the concentration of MB is doubled. The preparation method is similar to the method described in the example of CS for the influenza vaccine with A803 + ΜΡ ^ adjuvant, with the only difference being that the concentration of MBI is doubled.

Control: a full dose of P1iaph ™ by i / m administration.

Four planned visits for 1 subject: on the 0th, 21st, 90th and 180th days with a blood sample taken at each visit to assess immunogenicity.

Vaccination schedule: 1 injection of influenza vaccine on day 0.

ΧΙΙ. 3. Immunogenicity results.

ΧΙΙ.3.1. Endpoints and results for SM!

Primary Endpoint Assessment

On the 21st day: СΜI response in all regarding the frequency of occurrence of influenza-specific ί.Ό4 T-lymphocytes per 10 ⁶ in tests producing at least two different cytokines (Ш-2, ΙΡΝ-γ, Т \ Т ' -t and (Ό40Ι.).

To assess the CΜI response, the frequency of occurrence of virus-specific CE4 is analyzed as follows.

CM-Ratio regarding the frequency of occurrence of virus-specific CE4 between groups vaccinated with adjuvant vaccines and и1ιι-ΥΝ & is obtained using the ΛNСΟУА model with logarithmically converted titers. The ANСΟУА model includes a vaccine group as a fixed effect and a logarithmically converted titer before vaccination as a parameter of the regression equation. SM relationships and their 98.75% CIs result from exponential

- 65 011419 transformations of the corresponding group matching within the framework of this model. 98.75% CI for the corrected SM (geometric mean) is obtained as a result of the exponential conversion of 98.75% CI for the group mean value obtained by the least squares method within the framework of the ANSNUΆ model mentioned above.

Results: inferential analysis (table. 47)

Adjusted SM and CM ratios (with their 98.75% CI) specific for influenza virus C1) 4 T-lymphocytes producing at least two cytokines (^ -2, ΈΡΝ-γ, TBE-α and C1) 40Е) on The 21st day after repeated stimulation with 1p νίΐΐΌ combined antigens II are presented in table. 47. For each adjuvanted influenza vaccine, the upper limit of the bilateral (1 \\ 'o-kk1ssG) 98.75% CI SM-ratio is significantly lower than the clinical limit of 2.0. This demonstrates the equally effective efficacy of both adjuvanted influenza vaccines given to older subjects compared to the E1iaph ™ vaccine given to adults aged 18–40 years in terms of the frequency of occurrence of influenza-specific Cl1-4 viruses after vaccination.

Table 47 The adjusted CM ratio of influenza virus-specific C1) 4, producing at least two cytokines, on day 21 (ATP cohort for immunogenicity)

Corrected CM ratio (Ηιι-ΥΝα / Α803 + ΜΡΙ- (conc. 1)) Ηιι-ΥΝΟ AZOZ + ΜΡί. (conc. 1) 98.8% CI N The correction. OM N The correction. CM Value Ι Ι be 70 1995,3 72 2430.0 0.82 0.65 1,04

Corrected CM ratio (ΕΙιι-ΥΝΟ / Α803 + ΜΡΙ- (conc. 2)) ΕΙιι-ΥΝΟ Α803 + ΜΡΙ- (conc. 2) 98.8% CI Ν The correction. CM N The correction. CM Value and υι 70 1979,4 72 2603.8 0.76 0.59 0.98

Corrected CM = geometric mean for the antibody, adjusted relative to the background titer.

Ν = number of subjects with available results both before and after vaccination.

98.8% CI = 98.8% confidence interval for the adjusted SM relationship (Άηсονа model: background correction).

EE = lower limit, BB = upper limit.

Results: descriptive analysis (FIG. 26)

The following main results were obtained:

before CMI vaccination, the response is higher in young adults than in older people;

after vaccination, the enhancing effect of the influenza vaccine on the CMI response was observed in young adults (18-40 years old),

The CMI response in older adults who received the adjuvanted influenza vaccine is comparable to the CMI response in young adults;

the difference between the states before and after vaccination in the responses of SB4 T-lymphocytes for all studied cytokines (ΊΕ-2, SB40E, ΊΝΕ-α and ΖΕΝ-γ) was significantly higher when using adjuvanted vaccines compared with E1iapkh ™ (18-40 years old) ) for all tests, with the exception of the case when the inventors compare E1iapkh (18-40 years old) and E1ts / A803 + MPB (concentration 1).

It should be noted that 1p νп stimulation was carried out using Е1и strains: (1) B / Lapdka, (2) A / Η3N2 / Nсν of Wogk and (3) A / 113№ / Шуо1пту, instead of A / ШУ / Ba Са1собош, included into the vaccine. However, previous data, including the vaccine strain ΛΉ1Ν1: Να \ · Ca1bosha from a subgroup of subjects, indicate that the results will be similar.

Results: evaluation of the tertiary end point (table. 48)

In order to assess the tertiary endpoint, the frequency of occurrence of influenza-specific SB4 / SBV T-lymphocytes and memory B cells was measured on days 0, 21, 90 and 180.

The frequency of occurrence of influenza-specific cytokine-positive SB4 / SB8 T-lymphocytes for each antigen was summarized (descriptive statistics) for each vaccinated group on days 0 and 21.

A nonparametric test (Wilcoxon test) was used to compare the differences between the two groups (influenza adjuvant vaccine against E1iapx ™), and the statistical p value was calculated for each antigen in each other test.

Descriptive statistics on the individual differences between the responses on day 21 / day 0 (after / before vaccination) are calculated for each vaccinated group and each antigen in each other test.

To compare the individual differences after / before vaccination, a nonparametric criterion (Wilcoxon test) is used, and the statistical value of p will be calculated for each antigen in

- 66 011419 every other test.

The p values according to the Wilcoxon criterion used to compare differences in the frequency of occurrence of influenza-specific C1-4 T cells are presented in Table. 48.

Table 48

Conclusion statistics: p values according to the Kruskal-Wallis criteria for C1) 4 T cells

P value (ATP-ι in

by immunogenicity)

Group 1 and Yai-E1 O Group 2 and PJ Group 1 and Ηιι-ΥΝΘ Group 2 and Pi-ΥΝΘ Day 0 21st day Day 0 21st day Day 0 21st day Day 0 21st day ALL FOR TWO 0.4380 0,0003 0.4380 0,0003 0.0000 0.9014 0,0005 0.4889 Οϋ40Ι 0.3194 0,0002 0.3194 0,0002 0.0000 0.9841 0,0003 0.5412 ΙΝΕγ 0.5450 0,0004 0.5450 0,0004 0.0000 0.5397 0.0001 0.7895 11.2 0.3701 0,0008 0.3701 0,0008 0,0003 0.8557 0.0022 0.4766 ΤΝΕα 0.3716 0,0004 0.3716 0,0004 0.0000 0.8730 0.0013 0.2114

Group 1: Influenza vaccine with adjuvant A803-MPE (concentration 1).

Group 2: Influenza vaccine with adjuvant A803-MPE (concentration 2). P1i-E’P1) = P1i-vaccine for the elderly.

Main conclusions:

(a) before OM vaccination, the frequency of occurrence of specific for influenza virus CE) 4 was similar in all groups of elderly subjects, but it was the highest in adults aged 18-40 years;

(b) after vaccination (day 21), the frequency of occurrence of specific for influenza virus CE) 4 Tlymphocytes was similar in elderly subjects vaccinated with adjuvant vaccines and in adults aged 18-40 years vaccinated with P1iapkh ™;

(c) in elderly subjects after vaccination (day 21), the frequency of occurrence of specific for the influenza virus CE) 4 T-lymphocytes was significantly higher after vaccination with adjuvant vaccines than P1iapkh ™;

(d) OM the frequency of occurrence of specific for influenza virus CE) 8 T cells before vaccination and after vaccination was essentially the same in all groups.

Results: endpoint evaluation for humoral immune response Observed variables

On days 0, 21, 90 and 180: serum antibody hemagglutination (ΗΙ) inhibitory titers tested separately against each of the three influenza virus strains presented in the vaccine (anti-ISh ^ anti-Ι Ι3Ν2 and anti-B antibodies).

The reference value for the (titer) ΗΙ antibody against all vaccine antigens was determined in the laboratory before analysis (and it is 1:10). A seronegative subject is a subject whose antibody titer is lower than the reference value. A seropositive subject is a subject whose antibody titer is greater than or equal to the reference value. The antibody titer below the reference point in this analysis is represented by an arbitrary selected value equal to half the reference point.

Based on the titers of антител antibodies, the following parameters are calculated:

(a) geometric mean titers (OMT) of антител antibodies on days 0 and 21 calculated by taking the antilogarithm from the average for logarithmic titer conversions, (b) seroconversion factors (8P) on day 21, defined as multiple increase in serum OMT ΗΙ on day 21 compared with day 0, (c) seroconversion levels (8C) on day 21 as a percentage of vaccinees or with a либо titer before vaccination <1:10 and a titer after vaccination> 1 : 40, or with a titer before vaccination> 1:10 and at least a four-fold increase in titer after vaccination, (g) level seroprotection (8RK) at day 21 defined as the percentage of vaccinees with a serum ΗΙ-titer> 1:40.

95% CI for OM is obtained within each group individually. First, 95% CI is obtained for the average value of the logarithmically converted titer under the assumption that the logarithmically converted titers are characterized by a normal distribution with an unknown variable. Then get 95% CI for OM as a result of the exponential conversion of 95% CI for the average value of the log-transformed titer. The missing serological result for measuring a specific antibody is not replaced. Therefore, a subject without a serological result for a given time point does not contribute to the test analysis at that time point.

The results of the humoral immune response (Fig. 27 and table. 49)

OMT ΗΙ antibodies before vaccination for all three vaccine strains were in the same range in the four treatment groups. After vaccination, there is an obvious effect of two adjuvants, which increase the humoral response in the elderly compared to standard P1 and ap in the same population.

- 67 011419

SMT:

significantly higher with respect to Η1Ν1 for A803 + MBP (concentration 2), significantly higher with respect to Η3Ν2 and with respect to B for both adjuvants.

Twenty-one days after vaccination, P1iapx subjects (18–40 years old) had a higher Y response for Ca1dosh and B / Lapzi strains.

As shown in the table. 49, adjuvanted influenza vaccines exceeded the requirements of European authorities for the annual registration of cleaved virionic influenza vaccines OG appia1 81gat sRapdez (CPMR / WUR / 214/96)] for subjects over the age of 60 years.

After vaccination, there was a statistical difference in seroprotection levels of L antibodies between the P1iag1x group (over 65 years old) and P1i-A803 + MPb (concentration 2) for strain A / Νϋν Ca1edosh.

For each vaccine strain, the seroprotection levels for the two anti-influenza adjuvant vaccine groups are in the same range compared to the P1iapch group (18-40 years old).

There was a statistical difference in seroconversion levels of L antibodies between the P1iapch group (over 65) and

P1i-A803 + MPb (concentration 2) for the strain AMe ^ Ca1edosha,

P1i-A803 + MPB (concentration 1) for strain B / Lapji.

For each vaccine strain, seroconversion levels for the two anti-influenza adjuvant vaccine groups are in the same range compared to the P1iapkh group (18–40 years old), with the exception of the Ca1edosh strain.

Table 49

Seroprotection levels, seroconversion levels and conversion factors on the 21st day (ATP cohort by immunogenicity)

Strains Group N Seroprotection Level (ΗΙ-titer> 40)% Seroconversion Level (£ 4X) [95% CI] Conversion Factor [95% CI]% EU Standard (> 60 years old) > 60% > 30% > 2.0 Standard 1 EU (<60 years old) > 70% > 40% > 2.5 Α / Νβνν None Upd 75 100 [95.20-100] 77.3 [66.2-86.2] 35.1 [21.9-56.4] Ca1ec1op1 Pi EgbeNu 49 71.4 [56.74-83.42] 30.6 [18.3-45.4] 3.7 [2.4-5.7] a (Η1Ν1) PiA803 + MP1_ (conc. 1) 75 90.5 [81.48-96.11] 55.4 [43.4-67.0] 6.4 [4.5-9.0] PiA803 + MP! _ (Conc. 2) 75 98.7 [92.79-99.97] 74.7 [63.3-84.0] 9.2 (6.4-13.3] Α / Νβνν Pi Upd 75 93.3 [85.12-97.80] 76.0 (64.7-85.1] 9.2 (7.1-11.8] Wogk Pi EgbePu 49 81.6 [67.98-91.24] 69.4 (54.6-81.7] 8.2 (5.7-11.8] (Η3Ν2) PiA803 + MP1_ (conc. 1) 75 94.6 [86.73-98.51] 90.5 (81.5-96.1] 19.2 [14.6-25.3] PiA803 + MP1_ (conc. 2) 75 93.3 [85.12-97.80] 82.7 [72.2-90.4] 15.0 [11.2-20.2] W / Lapds Pi Upd 75 100 [95.20-100] 80.0 [69.2-88.4] 13.9 [10.1-19.1] and (B) None EGS! EG1U 49 93.9 [83.13-98.72] 81.3 [70.7-89.4] 4.3 (3.0-6.1] Pyazosis + MRI (conc. 1) 75 95.9 (88.61-99.16] 73.0 (61.4-82.6] 8.5 [6.5-11.2] Pyazosis + MRI (conc. 2) 75 98.7 [92.79-99.97] 66.7 [54.8-77.1] 7.6 [5.6-10.2]

Ν = total number of subjects.

% = percentage of subjects with a titer on day 21 within a specific range. CI = confidence interval.

HP. 3.2. Conclusions on immunogenicity.

(a) The incidence of influenza-specific CE4 before vaccination was significantly lower in older adults than in adults aged 18-40 years. After P1iapx ™ vaccination, the post-vaccination frequency of occurrence (day 21) remained lower in older adults compared to younger people. In contrast, no less effective in terms of the frequency of occurrence with respect to the post-vaccination frequency of the occurrence of influenza-specific C1-4 viruses after vaccination with adjuvanted vaccines in elderly subjects was demonstrated in comparison with the P1iapkh ™ vaccination in adults aged 18-40 years.

(b) With regard to the humoral immune response in terms of the N-antibody response, all influenza vaccines met the requirements of the European authorities for the annual registration of inactivated influenza vaccines ! 1e 1ttypo1o§1sa1 azzzztep! OG appia1 81gat sRapdez (СРМР / ВУР / 214/96)]. In older adults, adjuvant vaccines mediated at least a tendency to have a higher humoral immune response to influenza hemagglutinin than P1iapx ™. A significant difference between the humoral immune response against each vaccine strain is mediated

- 68 011419 ny in elderly subjects with adjuvant vaccines in comparison with P1iapkh ™, is summarized in table. 50. Compared with adults aged 18-40 years vaccinated with P1iapkh ™, elderly subjects vaccinated with adjuvant vaccines showed a tendency to higher post-vaccination OMT and seroconversion factor on the 21st day against strain A / No ^ Wogk.

Table 50

Significant difference in the humoral immune response between adjuvant vaccines and P1iapch in elderly subjects

With MT after vaccination Seroconversion factor Seroprotection level and Seroconversion rate E1iA503 + MP1_ (conc. 1) Α / Νβνν Wogk VLL Α / Νενν Wogk - Valapji E1iA503 + MP1_ (conc. 2) Α / Νβνν Wagk WALapdea Α / Νθνν Ca1eSop! A Α / Νβνν Ca1ebop1a Α / Νθνν Ca1ec1op1a Α / Νθνν Ca1ec1ogna

ΧΙΙ. 4. Reactogenicity results.

ΧΙΙ.4.1. Registration of adverse events (AE).

Concerned symptoms were recorded (see Table 51), occurring within 7 days of the follow-up period (vaccination day and 6 subsequent days). In addition, unexpected symptoms were recorded that occurred within 21 days of the observation period (vaccination day and 20 + 3 subsequent days). The intensity of the following AEs was evaluated as described in table. 52. Table 51

Disturbing local / general adverse events

Attention: temperature was recorded in the evenings. If necessary, additional temperature measurements at another time of the day recorded the highest temperature value.

Table 52

Intensity Scores for Anxiety Symptoms in Adults

Adverse event Intensity Rating Parameter Pain at the injection site 0 Missing one when touching 2 when moving with a limb 3 interferes with ordinary activities Redness at the injection site Record the largest surface size in mm Swelling at the injection site Record the largest surface size in mm Hematoma at the injection site Record the largest surface size in mm Fever* Record temperature in ° C / ° E Headache 0 Missing one easy to carry 2 affects ordinary activities 3 interferes with ordinary activities Fatigue 0 Missing one easy to carry 2 affects ordinary activities 3 interferes with ordinary activities Joint pain at the injection site and other places 0 Missing one easy to carry 2 affects ordinary activities 3 interferes with ordinary activities Muscle pain 0 Missing one easy to carry 2 affects ordinary activities 3 interferes with ordinary activities Shiver 0 Missing one easy to carry 2 affects ordinary activities 3 interferes with ordinary activities

A fever is determined if the temperature in the armpit is greater than or equal to 37.5 ° C (99.5 ° P).

- 69 011419

The maximum intensity of local redness / swelling at the injection site is evaluated as follows: 0 means 0 mm; 1 means from> 0 to <20 mm; 2 means from> 20 to <50 mm; 3 corresponds to> 50 mm. The maximum fever intensity is evaluated as follows: 1 means from> 37.5 to <38.0 ° C; 2 means from> 38.0 to <39.0 ° C; 3 means> 39.0 ° C.

The researcher assesses the intensity for all other AEs, i.e. unforeseen symptoms, including BAE (severe AE), recorded during the study. The assessment is based on the clinical experience of the researcher. The intensity of each recorded AE is evaluated in one of the following categories:

(weak) corresponds to AE, which is easily tolerated by the subject, leading to minimal discomfort and not affecting everyday activities;

(moderate) corresponds to AE, which is quite uncomfortable with regard to the impact on ordinary daily activities;

(severe) corresponds to AE, which interferes with normal daily activities (in adults / adolescents, such AE may, for example, create an obstacle to being at work / at school and force the introduction of corrective therapy).

HP. 4.2. Registration of adverse events (AE).

It was found that the reactogenicity observed in elderly subjects when using adjuvanted vaccines, in relation to both local and general symptoms, was higher than when using B1iapkh ™ in the same population. However, it was shown that this corresponds to the level shown for the adult population. The onset and intensity of symptoms increased after vaccination with adjuvant vaccines compared with the reactogenicity shown in elderly subjects using B1iapkh ™ (Fig. 28). In all cases, the symptoms quickly disappeared.

The highest symptom tendency with a score of 3 was shown in the group of subjects receiving the MPI adjuvanted vaccine at the highest concentration compared to the group of subjects receiving the adjuvanted vaccine in which MPI was present in a lower concentration. However, in all cases, the symptoms quickly disappeared.

Claims (60)

1. A multivalent anti-influenza immunogenic composition comprising an influenza virus antigen or antigenic preparation of at least two strains of influenza virus, where at least one strain is associated with an outbreak of a pandemic, or may be associated with an outbreak of a pandemic, in combination with an adjuvant in the form of an emulsion of the type oil-in-water, wherein said oil-in-water emulsion adjuvant contains a metabolizable oil, sterol and / or alpha-tocopherol and an emulsifier. 2. The composition according to claim 1, where the specified emulsion of the type oil-in-water contains droplets of oil, of which at least 70% in intensity have a diameter of less than 1 μm. 3. The composition according to claim 1 or 2, where the specified emulsion of the type oil-in-water contains droplets of oil, of which at least 70% in intensity have a diameter of less than 500 nm. 4. The composition according to any one of claims 1 to 3, where the specified oil-in-water emulsion contains droplets of oil, of which at least 80% in intensity have a diameter of less than 300 nm. 5. The composition according to any one of claims 1 to 4, where the specified oil-in-water emulsion contains droplets of oil, of which at least 90% in intensity have a diameter in the range of 120-200 nm. 6. The composition according to any one of claims 1 to 5, where the specified metabolizable oil is squalene. 7. The composition according to any one of claims 1 to 6, where the specified metabolizable oil is present in an amount of from 0.5 to 20% of the total volume of the specified immunogenic composition. 8. The composition according to any one of claims 1 to 7, where the specified metabolizable oil is present in an amount of from 1.0 to 10% of the total volume of the specified immunogenic composition. 9. The composition according to any one of claims 1 to 8, where the specified metabolizable oil is present in an amount of from 2.0 to 6.0% of the total volume of the specified immunogenic composition. 10. The composition according to any one of claims 1 to 9, wherein said sterol is cholesterol. 11. The composition according to any one of claims 1 to 10, where the specified alpha-tocopherol is present in an amount of from 1.0 to 20% of the total volume of the specified immunogenic composition. 12. The composition according to any one of claims 1 to 11, where the specified alpha-tocopherol is present in an amount of from 1.0 to 5.0% of the total volume of the specified immunogenic composition. 13. The composition according to any one of claims 1 to 12, where the ratio of squalene: alpha-tocopherol is equal to or less than 1. 14. The composition according to any one of claims 1 to 13, where the specified emulsifier is a tween 80. 15. The composition according to any one of claims 1 to 14, where the specified emulsifier is present in an amount of from 0.01 to 5.0% (wt./wt.) By weight of the specified immunogenic composition. 16. The composition according to any one of claims 1 to 15, where the specified emulsifier is present in an amount of 0.12.0% (wt./wt.) By weight of the specified immunogenic composition. - 70 011419 17. The composition according to any one of claims 1 to 16, where the immunogenic composition further comprises a TK-4 ligand (To11-like receptor-4). 18. The composition according to any one of claims 1 to 17, wherein said TBK-4 ligand is selected from the list consisting of a non-toxic derivative of lipid A, such as 3L-MPB (3-de-O-acylated monophosphoryl lipid A); a synthetic derivative of lipid A; MPP (muramyl dipeptide) and protein P from K8U (respiratory syncytial virus). 19. The composition according to any one of claims 1 to 18, where the specified derivative of lipid A is 3ΌMRP. 20. The composition according to any one of claims 1 to 19, where 3L-MPI is present in an amount of 1-100 μg (wt./about.) Per dose of the composition. 21. The composition according to any one of claims 1 to 20, where 3L-MPI is present in an amount of 10-50 μg / ml. 22. The composition according to any one of claims 1 to 20, where 3b-MPI is present in an amount of about 25 μg / ml. 23. The composition according to any one of claims 1 to 22, wherein said polyvalent composition is a divalent, trivalent or tetravalent composition. 24. The composition according to any one of claims 1 to 23, where the specified pandemic strain of the influenza virus is selected from the list consisting of H5№, H9№, No. N7, No. N2 and NSh1. 25. The immunogenic composition according to paragraph 24, where at least two strains of the influenza virus are selected from the list consisting of H5№, H9№, No. N7, No. N2 and NSh1. 26. The immunogenic composition according to any one of claims 1 to 25, wherein said antigen or antigenic composition contains from 1 to 15 μg of HA (hemagglutinin) per strain of influenza virus. 27. The immunogenic composition of claim 26, wherein said antigen or antigenic composition comprises a low dose of HA per influenza strain. 28. The immunogenic composition of claim 27, wherein said antigen or antigenic composition comprises 2.5 to 7.5 μg HA per strain. 29. The immunogenic composition according to any one of claims 1 to 28, wherein the antigen or antigenic composition exists in the form of a purified whole influenza virus, an inactivated influenza virus, or a subunit (s) of the influenza virus component (s). 30. The immunogenic composition according to clause 29, where the specified inactivated influenza virus is a split influenza virus. 31. A method of preparing an influenza immunogenic composition for a pandemic situation, comprising mixing an influenza virus antigen or an antigenic preparation of at least two influenza virus strains, at least one of which is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic, with an oil type emulsion -in water. 32. The method according to p, where the adjuvant in the form of an emulsion of the type oil-in-water is as defined in any one of claims 1 to 16. 33. The method according to p, where the emulsion of the type oil-in-water further comprises an adjuvant TK-4. 34. The method according to p, where the adjuvant TK-4 is as defined in any one of paragraphs.17-22. 35. The immunogenic composition according to any one of claims 1 to 30 for use in medicine. 36. The use of (a) an influenza virus or its antigenic preparation and (b) an adjuvant in the form of an oil-in-water emulsion in the manufacture of an immunogenic composition according to any one of claims 1-30 for inducing at least one of the following: 1) enhanced 0-4 T-cell immune response, 2) enhanced Cellular secondary immune response against the specified virus or antigenic composition in humans. 37. The use of influenza virus or its antigenic preparation and adjuvant in the form of an oil-in-water emulsion in the manufacture of an immunogenic composition according to any one of claims 1-30 for vaccinating elderly people against influenza. 38. The application of clause 37, where the composition induces at least one of the following: 1) an enhanced 0-4 T-cell immune response, 2) an enhanced B-cell secondary immune response against the specified virus or antigenic composition in the specified elderly subject. 39. The use according to any one of claims 36-38, wherein administration of said immunogenic composition induces both an enhanced 0-4 T-cell immune response and an enhanced B-cell secondary immune response. 40. The use according to any one of claims 36 and 38, 39, wherein said 0-4 T cell immune response includes the induction of a cross-reactive 0. 4 T helper response. 41. The use according to any one of claims 36-40, wherein the age of the vaccinated population is more than 50 years. 42. The application of paragraph 41, where the age of the vaccinated elderly population is more than 65 years. 43. The use of influenza virus or its antigenic preparation in the manufacture of an immunogenic composition for the revaccination of people previously vaccinated with an immunogenic composition according to any one of claims 1 to 30. 44. The use of claim 43, wherein the composition that was used for the booster vaccine contains an additional adjuvant. - 71 011419 45. The application of claim 44, wherein said adjuvant is selected from the list consisting of an adjuvant in the form of an oil-in-water emulsion, an aluminum adjuvant, a TBK-4 ligand, and saponin. 46. The application of claim 45, wherein said adjuvant in the form of an oil-in-water emulsion is as defined in any one of claims 1-16, and the TK-4 ligand is as defined in any of claims 17 -22. 47. The use according to any one of claims 43-46, wherein said immunogenic revaccination composition comprises an influenza virus or antigenic preparation thereof that has common CE4 T cell epitopes with a cleaved influenza virus or a cleaved virus antigenic preparation used for the first vaccination. 48. The use according to any one of paragraphs 43-47, where the immunological response after revaccination is any one or two or all of the following: enhanced CO4 response against influenza virus or its antigenic drug, or enhanced humoral response, or enhanced B-cell secondary immune response. 49. The use according to any one of claims 43-48, wherein the influenza virus antigen or antigenic preparation thereof originates from at least two different strains of the influenza virus. 50. The use of claim 49, wherein the influenza virus antigen or antigenic preparation thereof comes from three different strains of the influenza virus. 51. The use according to any one of claims 43-50, wherein said immunogenic revaccination composition comprises at least one strain of influenza virus that is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. 52. The application of claim 51, wherein said pandemic strain is selected from the list consisting of Η5Ν1, Η9Ν2, Η7Ν7, Η2Ν2 and Η1Ν1. 53. The use according to any one of paragraphs 43-52, where the first vaccination is carried out with an influenza composition containing a strain of influenza virus that could potentially cause an outbreak of a pandemic, and revaccination is carried out with an influenza composition containing a circulating pandemic strain. 54. The use according to any one of claims 43 to 53, wherein said immunogenic composition comprises a low dose of HA antigen. 55. The use according to any one of paragraphs 43-54, wherein said influenza virus antigen or antigenic preparation thereof is obtained from chicken embryos or tissue cultures. 56. The use of an antigen or antigenic preparation from the first strain of influenza virus in the manufacture of an immunogenic composition according to any one of claims 1 to 30 for protection against influenza infections caused by a variant strain of influenza virus. 57. The application of claim 56, wherein the first strain of influenza virus is associated with an outbreak of a pandemic, or may be associated with an outbreak of a pandemic. 58. The use of claim 56, wherein the variant strain of influenza virus is associated with an outbreak of a pandemic or may be associated with an outbreak of a pandemic. 59. The application of claim 57 or 58, wherein said pandemic strain is selected from the list consisting of Η5Ν1, Η9Ν2, Η7Ν7, Η2Ν2 and Η1Ν1. 60. The use according to any one of claims 43-59, wherein said influenza virus antigen is selected from the list consisting of split influenza virus, whole influenza virus, subunit influenza virus, influenza virosome and its antigenic preparation.